http://2008.igem.org/wiki/index.php?title=Special:Contributions/BNathalie&feed=atom&limit=50&target=BNathalie&year=&month=2008.igem.org - User contributions [en]2024-03-28T15:16:02ZFrom 2008.igem.orgMediaWiki 1.16.5http://2008.igem.org/Team:KULeuven/17_September_2008Team:KULeuven/17 September 20082008-10-30T01:48:03Z<p>BNathalie: </p>
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== Lab Work ==<br />
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=== Wet Lab ===<br />
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We made a miniprep of the electroporations that succeeded: [http://partsregistry.org/Part:BBa_J23109 J23109]+[http://partsregistry.org/Part:BBa_J23078 J23078], [http://partsregistry.org/Part:BBa_K145253 K145253]+[http://partsregistry.org/Part:BBa_K145254 K145254], [http://partsregistry.org/Part:BBa_K145014 K145014] and [http://partsregistry.org/Part:BBa_K145201 K145201].<br />
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We started the analysis of the degradation of GFP-LVA for the thirth time, but this time with excellent results. Yesterday, we made liquid cultures of GFP, GFP-LVA and pUC. We re-inoculated them into fresh LB medium this morning and allowed the cells to grow for about 4 hours - this to make sure that the cells were in the log-phase when we start the measurements. Then we transferred the cells into a minimal ABT medium. This medium does not contain sugars or any other source of carbon. This way the cells will stop producing GFPs and so we can measure degradation. From the moment that we transferred the cells into this ABT medium, we started to measure the fluorescence of the cells with a FACS apparatus. We measured the fluorescence every 15 to 20 minutes for about 4 hours. The results were promising. The GFP with LVA tag showed a strong decline in fluorescence (as we expected) and the normal GFP was still very fluorescent after 4 hours.<br />
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=== Dry Lab ===<br />
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Started to make the lay-out of the notebook a little bit better.</div>BNathaliehttp://2008.igem.org/Team:KULeuven/16_September_2008Team:KULeuven/16 September 20082008-10-30T01:47:47Z<p>BNathalie: </p>
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== Lab Work ==<br />
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=== Wet Lab ===<br />
We started the day as we always did - we miniprepped some parts: K145175, I712074+J23078 and C0062+B0015. We also wanted to digest these parts, but we ran out of ''Spe''I. Actually we thought it would be futile to digest them, as the courses start next week and we will not have time to finish new ligations.<br />
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We had colonies of the electroporated cells. We did a PCR to test if these cells contained the correct (i.e. ligated) plasmid. K145014+pSB1A2 was definitely alright and K145201, J23109+J23078 and K145253+K145254 seemed OK as well (K145150+B0034+C0062 failed).<br />
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We also tried to redo the analysis of the degradation of GFP. At first we didn't detect any fluorescence. So we re-inoculated the cells into fresh LB medium and allowed them to grow for 4 hours. This made sure that the culture was still in de log-phase when we measured fluorescence. After these 4 hours we could detect fluorescence, but we lost our minimal ABT medium and so we couldn't test any further. The final attempt will follow tomorrow!<br />
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As we approach the end of our lab work, we also prepared some samples that will be sequenced. This way we can see if our ligations succeeded. The plasmids that are shown to be correct, will then be officially submitted to the registry.</div>BNathaliehttp://2008.igem.org/Team:KULeuven/15_September_2008Team:KULeuven/15 September 20082008-10-30T01:47:28Z<p>BNathalie: </p>
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== Lab Work ==<br />
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=== Wet Lab ===<br />
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We wanted to test whether our GFP with LVA tag is degraded faster than a normal GFP (using FACS). But for some reason the FACS apparatus didn't detect any fluorescence in our liquid culture of GFP-LVA. The colonies on the plates, however, were still fluorescent. We will try again tomorrow.<br />
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We also did a miniprep of J23116+B0034 and we tried to digest it with ''Spe''I and ''Eco''RI, but the digest failed. It was the last drop of ''Spe''I and we presume that this enzym didn't work anymore.<br />
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We had colonies on all the parts that were electroporated yesterday (but not very much). We did a PCR test to see if they were ligated correctly and it seemed to be OK (C0062+B0015, I712074+J23078 and K145275).<br />
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Apart from that, we also electroporated yesterday's ligation mixtures: K145014+pSB1A2, J23109+J23078, K145253+K145254, J23116+K145201, J23116+B0032+K145201 and (K145150+B0034)+C0062.</div>BNathaliehttp://2008.igem.org/Team:KULeuven/14_September_2008Team:KULeuven/14 September 20082008-10-30T01:47:10Z<p>BNathalie: </p>
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== Lab Work ==<br />
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=== Wet Lab ===<br />
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We occupied ourself in the lab with the usual stuff:<br />
* Miniprep: K145257.<br />
* Electroporation: C0062+B0015, I712074+J23078 and K145275.<br />
* Digests: K145257 was cut with ''Eco''RI and ''Xba''I, yesterday's digests were continued: <div style="margin-left:10px;"> cut with ''Pst'' I -> J23109, K145150+B0034 and K145013. </div> <div style="margin-left:10px;"> cut with ''Eco''R I -> K145272, K145013+B0015, K145001+B0015, K145254 and K145201. </div> <br />
* PCR to test electroporations of yesterday: We had colonies of J23116+B0034 and the PCR test showed that it was ligated properly.<br />
* Ligation mix: K145014+pSB1A2, J23109+J23078, K145253+K145254, J23116+K145201, J23116+B0032+K145201 and (K145150+B0034)+C0062.</div>BNathaliehttp://2008.igem.org/Team:KULeuven/13_September_2008Team:KULeuven/13 September 20082008-10-30T01:46:53Z<p>BNathalie: </p>
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== Lab Work ==<br />
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=== Wet Lab ===<br />
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[[image:T7kopie.jpg|right|thumb|380px|agarose gel with the PCR samples of T7 DNA polymerase with UmuD tag (BBa_K145014)]]<br />
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We miniprepped two cultures today: K145272 and K145013+B0015.<br />
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We also did a new PCR test on the electroporations of the day before yesterday (but different colonies). We found that colony 3 of B0014+B0033+K145151+B0015 is probably correct. The other ones were still wrong though.<br />
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We did step 2 of the PCR to construct T7 DNA polymerase with UmuD tag - but without purifying the mix obtained in step 1. And check it out, it seemed to have worked! Hurrah! Take a look at the picture on the right and you will see that we had a band at the correct height (2750 bp). Thank you professor Robben.<br />
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Apart from that, we also electroporated J23116+B0034 and K145275; and we made a ligation mix of C0062+B0015 and I712074+J23078.<br />
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We set up some digests. After 2 hours we put them on gel to see if they were properly cut. Unfortenately they weren't. We let them stand overnight and hope that they will be digested by tomorrow (it were the last bits of enzym, so maybe they don't work anymore): <br />
<div style="margin-left:100px;"> cut with ''Spe'' I -> J23109, K145150+B0034 and K145013. </div> <div style="margin-left:100px;"> cut with ''Xba'' I -> K145272, K145013+B0015, K145001+B0015, K145254 and K145201. </div> <div style="margin-left:100px;"> cut with ''Eco''R I and ''Spe'' I -> K145014 (T7-UmuD). </div></div>BNathaliehttp://2008.igem.org/Team:KULeuven/12_September_2008Team:KULeuven/12 September 20082008-10-30T01:46:35Z<p>BNathalie: </p>
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== Lab Work ==<br />
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=== Wet Lab ===<br />
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Last Friday, we had a lot of good news. This time it is bad news :-(<br />
* There were no colonies on the plates of three ligations that we electroporated yesterday (J23116+B0032+C0062+B0015, K145253+ K145254 and J23109+J23032+K145001+B0015). We did a PCR test to see whether the other ones were properly ligated. Only one of them seemed alright (K145272). <br />
* We miniprepped some cultures: R0040+J23066+J23109+J23032 and K145278.<br />
* The end-filling reaction to make J23078 was put on gel. It appeared to be OK.<br />
* We made a number of digests: <div style="margin-left:10px;"> cut with ''Eco''RI and ''Spe''I -> R0040+J23066+J13109+J23032 (succeeded). </div> <div style="margin-left:10px;"> cut with ''Xba''I and ''Eco''R I -> K145278 (failed). </div> <div style="margin-left:10px;"> cut with ''Xba''I and ''Pst'' I -> J23078 (PCR product). </div> <div style="margin-left:10px;"> cut with ''Spe''I and ''Pst'' I -> C0062, J23109 and I712074 (J23109 failed). </div><br />
* We set up two more ligations: J23116+B0034 and K145275.<br />
* We also continued our quest for T7 polymerase with UmuD tag. It was suggested that the problem may lie in the fact that we use a gel extract as a template (there might still be a lot of agarose in the sample). Therefore we started step 1 of the PCR (first halve of the tag). We will do step 2 of the PCR (second halve of the tag) tomorrow with the unpurified PCR mix of step 1 as template.<br />
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=== Dry Lab ===<br />
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Actual KULeuven iGEM logo redo<br />
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==== Modeling ====<br />
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Really finalising and closing (amongst others) the sensitivity books forgood :)</div>BNathaliehttp://2008.igem.org/Team:KULeuven/11_September_2008Team:KULeuven/11 September 20082008-10-30T01:46:18Z<p>BNathalie: </p>
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== Lab Work ==<br />
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=== Wet Lab ===<br />
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We electroporated the following parts: K145201+(R0040+P0353), C0062+(K145150+B0034), K145253+K145254, (I712074+J23032)+(E0040+B0015), (B0014+B0033)+(K145151+B0015), (J23116+B0032)+(C0062+B0015), (J23109+J23032)+(K145001+B0015) and B0015+K145013.<br />
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We made a new ligation mix: J23116+B0034.<br />
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We made some digests with ''Spe''I: J23109 and I712074.<br />
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The oligos to make the new lock arrived today. So we started the end-filling procedure to make this part (J23078) using Klenow polymerase.<br />
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We also started to measure our input-output systems (K145279 and K145280) using FACS. Normally, when no anhydrotetracyclin is present, the cells shouldn't fluoresce. Unfortunately, the did already fluoresce from the start. We added anhydrotetracyclin (50 ng/ml) to the liquid cultures and allowed the cells to grow in this aTc medium for 4 hours. Then we measured the fluorescence again. Ideally, adding aTc would switch on the fluorescence. So we expected that there would be more fluorescence. This was not the case, we measured just as much fluorescence as in the beginning. ''The results can be found in the figures below''.<br />
Because the cells always fluoresce, the outputs definitely work. But we can not switch them on or off. This means that there is something wrong with the input. Maybe it wasn't properly ligated (-> sequencing)? Maybe there is not enough repressor (-> stronger RBS)? Maybe the repressor doesn't work (-> big problem)?<br />
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[[image:FACS-K145279.jpg|left|thumb|377px|'''FACS of K145279 (GFP without tag)''': purple surface = before adding aTc; green line = after adding aTc]] <br />
[[image:FACS-K145280b.jpg|right|thumb|377px|'''FACS of K145280 (GFP with LVA tag)''': purple surface = before adding aTc; green line = after adding aTc]]<br />
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=== Dry Lab ===<br />
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==== Modeling ====<br />
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Finalising and rounding up. Still messing with the sensitivity analysis...</div>BNathaliehttp://2008.igem.org/Team:KULeuven/10_September_2008Team:KULeuven/10 September 20082008-10-30T01:45:56Z<p>BNathalie: </p>
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== Lab Work ==<br />
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=== Wet Lab ===<br />
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* We MiniPrepped the ligations that succeeded yesterday: K145201+K145205, K145201+R0040+E0240, R0040+B0033+C0056+B0015. Then K145258 was cut with ''EcoR''I and ''Spe''I.<br />
* We also made a glycerolstock of these ligations.<br />
* The digests we made yesterday were put on gel. Then we extracted them and put again 2ul on gel to estimate the concentration.<br />
* We ligated the following parts: K145201 + (R0040+P0353, C0062 (Pst) + (K145150+B0034, K145253 + K145254, (I712074+J23032) + (E0040+B0015), (B0014+B0033) + (K145151+B0015), B0015 (Pst) + (K145013+pSB1A2). <br />
* We cut B0034 and J23109+J23032 with ''Eco''RI and ''Xba''I.<br />
* We made a liquid culture of K145279 and K145280 to test the relation input/output with anhydrotetracycline.<br />
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=== Dry Lab ===<br />
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Blending the ethics...<br />
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== Remarks ==<br />
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FC André had an important match today, the final score was 8-8! We were there and it was clear our team was far better than the others! They diserved to win!!!!</div>BNathaliehttp://2008.igem.org/Team:KULeuven/8_September_2008Team:KULeuven/8 September 20082008-10-30T01:45:28Z<p>BNathalie: </p>
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== Lab Work ==<br />
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=== Wet Lab ===<br />
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Final schemes:<br />
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[[Image:8sept final.PNG|thumb|left|370px]]<br />
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[[Image:8sept parallel.PNG|thumb|right|383px]]<br />
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=== Dry Lab ===<br />
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==== Wiki ====<br />
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IE fixes, for the rest of the day, I tried to hack the guestbook so the links didn't appear at the bottom, but nothing at all worked.</div>BNathaliehttp://2008.igem.org/Team:KULeuven/7_September_2008Team:KULeuven/7 September 20082008-10-30T01:43:52Z<p>BNathalie: </p>
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== Lab Work ==<br />
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=== Wet Lab ===<br />
Another day, another lab report:<br />
* We did a PCR of the hybrid promoter with the prefix-suffix primers. This gave about the same results as yesterday, albeit a bit better. Maybe because we annealed at a higher temperature this time. Anyhow, we still saw a bit of laddering which we presume to be caused by concatenation of the prefix-suffix primer into constructs like (- Prefix - Promoter - Suffix - )<sub>X</sub>. We cut out the (quite broad) band at the right length and extracted the DNA. We hope that by digesting, we once again get the decent cut-out part.<br />
* We made a gel with the PCR samples of the ligation test and with the digests (K145201, K145205, K145253, K145251 and R0040+J23022+J23109+J23032). The result was very good, we now have a lot of new BioBricks that are finished. Only R0040+J23022+J23109+J23032 gave bad results. Both the PCR and the digest was too short. This is the second time that this ligations fails. We think the problem lies in the first part (R0040+J23022). We will not use this one anymore. We wil use R0040+J23066 instead. This ligation definitely succeeded and this part contains key3d and is compatible with the old lock (J23032).<br />
* Yet another failure: PCR of T7 polymerase with UmuD tag failed again (surprise!). We followed the protocol that gave us 3 bands earlier but this time we did only 2 annealing steps at 56 degrees in stead of 3. The third step was replaced by one at 62 followed by 32 at 68 degrees.<br />
* We also found out that the part R1052 doesn't contain a prefix or suffix. That's why the digest and PCR didn't work. We will make some oligos to construct it ourself.<br />
* We electroporated the following parts: K145150+pSB1A2, K145150+E0240, K145150+B0034, K145013+pSB1A2, R0040+P0353, E0040+B0015, B0014+B0033+C0061+B0015 and K145254.<br />
* Set up some ligations: K145201+K145205, K145201+(R0040+E0240), (R0040+B0033)+(C0056+B0015), (R0040+J23066)+(J23109+J23032), (J23109+J23032)+(K145001+B0015) and (J23116+B0032)+(C0062+B0015).<br />
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So, we now have a few new BioBricks. A list of all the BioBricks that should be finished by the end of this month (including those needed for tests and intermediates), can be found [https://2008.igem.org/Team:KULeuven/Ligation here]. You can also see which ones have succeeded so far.<br />
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=== Dry Lab ===<br />
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Designed 2 oligos to construct R0052 (replacing R1052) ourselves. Will enter them as soon as possible.<br />
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== Comic of the day ==<br />
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<div class="center">http://imgs.xkcd.com/comics/purity.png</div></div>BNathaliehttp://2008.igem.org/Team:KULeuven/6_September_2008Team:KULeuven/6 September 20082008-10-30T01:41:51Z<p>BNathalie: </p>
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== Lab Work ==<br />
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=== Wet Lab ===<br />
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Busy day for a saturday ;)<br />
* Made some new electrocompetent TOP10 cells, we've got about 32 new aliquots of bacteria happy to be fried by a thousand volts.<br />
* PCR with prefix-sufix primers on [http://partsregistry.org/Part:BBa_R1052 R1052] and the hybrid promoter. Gave some weird results. Will need to be looked at again.<br />
* Made a couple digests: [http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_E0240 E0240], [http://partsregistry.org/Part:BBa_C0056 C0056]+[http://partsregistry.org/Part:BBa_B0015 B0015] and [http://partsregistry.org/Part:BBa_C0061 C0061]+[http://partsregistry.org/Part:BBa_B0015 B0015] were cut with ''Eco''RI, E0040 was cut with ''Spe''I and ''Pst''I, and [http://partsregistry.org/Part:BBa_B0015 B0015] was cut with ''Xba''I and ''Pst''I. They were put on gel and then purified. The results looked very good.<br />
* Also made a digest of [http://partsregistry.org/Part:BBa_K145201 K145201], [http://partsregistry.org/Part:BBa_K145205 K145205], [http://partsregistry.org/Part:BBa_K145253 K145253], [http://partsregistry.org/Part:BBa_K145251 K145251] and [http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_J23022 J23022]+[http://partsregistry.org/Part:BBa_J23109 J23109]+[http://partsregistry.org/Part:BBa_J23032 J23032], but then I forgot to load the Smartladder onto the gel. These digest were set up again (overnight reaction). Anyway, the gel already looked very promising ;-)<br />
* Started some new ligations (mainly to test the new hybrid promoter): R0040+P0353, (R0040+B0032)+(C0062+B0015), (R0011+B0032)+(C0061+B0015) and E0040+B0015.<br />
* We set up a PCR to test our ligations using the VF2/VR primers. <br />
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== Comic of the day ==<br />
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<div class="center">http://www.phdcomics.com/comics/archive/phd090308s.gif</div></div>BNathaliehttp://2008.igem.org/Team:KULeuven/5_September_2008Team:KULeuven/5 September 20082008-10-30T01:35:58Z<p>BNathalie: </p>
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== Lab Work ==<br />
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=== Wet Lab ===<br />
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We had a very happy day in the lab today and this is what we did:<br />
* We put the digests of [http://partsregistry.org/Part:BBa_R0011 R0011]+[http://partsregistry.org/Part:BBa_B0033 B0033], [http://partsregistry.org/Part:BBa_K145001 K145001]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_K145150 K145150] and [http://partsregistry.org/Part:BBa_K145013 K145013] on gel. They seemed OK and so we extracted them out of the gel.<br />
* We made a miniprep and digest of the following parts: <div style="margin-left:10px;"> cut with ''Eco''RI and ''Spe''I -> [http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_B0033 B0033], [http://partsregistry.org/Part:BBa_B0014 B0014]+[http://partsregistry.org/Part:BBa_B0033 B0033], [http://partsregistry.org/Part:BBa_R0053 R0053]+[http://partsregistry.org/Part:BBa_P0152 P0152], [http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_J23066 J23066] and [http://partsregistry.org/Part:BBa_R1052 R1052]. </div> <div style="margin-left:10px;"> cut with XbaI -> [http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_E0240 E0240], [http://partsregistry.org/Part:BBa_C0056 C0056]+[http://partsregistry.org/Part:BBa_B0015 B0015] and [http://partsregistry.org/Part:BBa_C0061 C0061]+[http://partsregistry.org/Part:BBa_B0015 B0015]. </div><br />
* We made a glycerolstock of the ligations that have suceeded so far [http://partsregistry.org/Part:BBa_C0040 C0040]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_C0060 C0060]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_C0012 C0012]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_K145015 K145015]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_E0240 E0240], [http://partsregistry.org/Part:BBa_K145151 K145151]+[http://partsregistry.org/Part:BBa_B0015 B0015].<br />
* We set up the following ligations: [http://partsregistry.org/Part:BBa_K145150 K145150]+[http://partsregistry.org/Part:pSB1A2 pSB1A2], [http://partsregistry.org/Part:BBa_K145150 K145150]+[http://partsregistry.org/Part:BBa_E0240 E0240], [http://partsregistry.org/Part:BBa_K145150 K145150]+[http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_K145013 K145013]+[http://partsregistry.org/Part:pSB1A2 pSB1A2] and (B0014+B0033)+(K145151+B0015).<br />
* Unfortunately, there were also some failures. The PCR for T7 polymerase with UmuD tag failed again. The result of the PCR test of the transduction was also a bit dodgy and the digest of R1052 resulted in a smear (but apparently it is a crappy part). A final attempt to construct T7 polymerase with UmuD tag was started (PCR).<br />
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But the most exciting thing today, were our flourescent colonies! We constructed two OUTPUT-subsystems containing GFP with and without LVA tag, as can be seen in this picture. On the left side GFP with LVA tag (our part K145015): less fluorescent. On the right side GFP without LVA tag (part E0240): more fluorescent. This means that our output and GFP-LVA work - hurrah!<br />
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[[Image:DSC02957.JPG|center|800px]]<br />
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Final Schemes so far:<br />
<br />
[[Image:5sept final.PNG|left|thumb|370px]]<br />
[[Image:5sept parallel.PNG|right|thumb|385px]]<br />
<br><br />
<br />
<br />
<br />
=== Dry Lab ===<br />
<br />
Dr. Coli and his danger, ethics, ethics, ethics...<br />
<br />
==== Modeling ====<br />
<br />
SimBiology2Latex Toolbox has been finalized and can be found on the wiki.<br />
<br />
MultiCell Toolbox has entered it's final design stages. A preview of the GUI can be found on the wiki.<br />
<br />
Some more work on diffusion has been done. Sensitivity Analyses is still a pain in the ***.<br />
<br />
==== Wiki ====<br />
<br />
Homepage has been revamped, removing a lot of bugs. IE fixes still need to follow. Components bar has been fixed.</div>BNathaliehttp://2008.igem.org/Team:KULeuven/5_September_2008Team:KULeuven/5 September 20082008-10-30T01:35:07Z<p>BNathalie: </p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
{{:Team:KULeuven/Tools/New_Day/Date_Retriever}}<br />
<br />
== Lab Work ==<br />
<br />
=== Wet Lab ===<br />
<br />
We had a very happy day in the lab today and this is what we did:<br />
* We put the digests of [http://partsregistry.org/Part:BBa_R0011 R0011]+[http://partsregistry.org/Part:BBa_B0033 B0033], [http://partsregistry.org/Part:BBa_K145001 K145001]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_K145150 K145150] and [http://partsregistry.org/Part:BBa_K145013 K145013] on gel. They seemed OK and so we extracted them out of the gel.<br />
* We made a miniprep and digest of the following parts: <div style="margin-left:10px;"> cut with ''Eco''RI and ''Spe''I -> [http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_B0033 B0033], [http://partsregistry.org/Part:BBa_B0014 B0014]+[http://partsregistry.org/Part:BBa_B0033 B0033], [http://partsregistry.org/Part:BBa_R0053 R0053]+[http://partsregistry.org/Part:BBa_P0152 P0152], [http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_J23066 J23066] and [http://partsregistry.org/Part:BBa_R1052 R1052]. </div> <div style="margin-left:10px;"> cut with XbaI -> [http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_E0240 E0240], [http://partsregistry.org/Part:BBa_C0056 C0056]+[http://partsregistry.org/Part:BBa_B0015 B0015] and [http://partsregistry.org/Part:BBa_C0061 C0061]+[http://partsregistry.org/Part:BBa_B0015 B0015]. </div><br />
* We made a glycerolstock of the ligations that have suceeded so far [http://partsregistry.org/Part:BBa_C0040 C0040]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_C0060 C0060]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_C0012 C0012]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_K145015 K145015]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_E0240 E0240], [http://partsregistry.org/Part:BBa_K145151 K145151]+[http://partsregistry.org/Part:BBa_B0015 B0015].<br />
* We set up the following ligations: [http://partsregistry.org/Part:BBa_K145150 K145150]+[http://partsregistry.org/Part:pSB1A2 pSB1A2], [http://partsregistry.org/Part:BBa_K145150 K145150]+[http://partsregistry.org/Part:BBa_E0240 E0240], [http://partsregistry.org/Part:BBa_K145150 K145150]+[http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_K145013 K145013]+[http://partsregistry.org/Part:pSB1A2 pSB1A2] and (B0014+B0033)+(K145151+B0015).<br />
* Unfortunately, there were also some failures. The PCR for T7 polymerase with UmuD tag failed again. The result of the PCR test of the transduction was also a bit dodgy and the digest of R1052 resulted in a smear (but apparently it is a crappy part). A final attempt to construct T7 polymerase with UmuD tag was started (PCR).<br />
<br />
But the most exciting thing today, were our flourescent colonies! We constructed two OUTPUT-subsystems containing GFP with and without LVA tag, as can be seen in this picture. On the left side GFP with LVA tag (our part K145015): less fluorescent. On the right side GFP without LVA tag (part E0240): more fluorescent. This means that our output and GFP-LVA work - hurrah!<br />
<br />
[[Image:DSC02957.JPG|center|800px]]<br />
<br />
<br />
<br />
Final Schemes so far:<br />
<br />
[[Image:5sept final.PNG|left|thumb|370px]]<br />
[[Image:5sept parallel.PNG|right|thumb|385px]]<br />
<br />
<br />
<br />
=== Dry Lab ===<br />
<br />
Dr. Coli and his danger, ethics, ethics, ethics...<br />
<br />
==== Modeling ====<br />
<br />
SimBiology2Latex Toolbox has been finalized and can be found on the wiki.<br />
<br />
MultiCell Toolbox has entered it's final design stages. A preview of the GUI can be found on the wiki.<br />
<br />
Some more work on diffusion has been done. Sensitivity Analyses is still a pain in the ***.<br />
<br />
==== Wiki ====<br />
<br />
Homepage has been revamped, removing a lot of bugs. IE fixes still need to follow. Components bar has been fixed.<br />
<br />
== Remarks ==<br />
<br />
== Strip of the day ==</div>BNathaliehttp://2008.igem.org/Team:KULeuven/4_September_2008Team:KULeuven/4 September 20082008-10-30T01:18:16Z<p>BNathalie: </p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
{{:Team:KULeuven/Tools/New_Day/Date_Retriever}}<br />
<br />
== Lab Work ==<br />
<br />
=== Wet Lab ===<br />
The ligations that were electroporated yesterday, gave a lot of colonies. One of these ligations (R0040+E0240) even resulted in fluorescent colonies. So this one definitely works (hurray!). We tried to test the other ligations with PCR, but we didn't use enough DNA.<br />
<br />
We loaded the PCR samples of yesterday onto an agarose gel. K145150 (hybrid promoter) and K145013 (antisense LuxI) gave nice results. K145014 (T7-UmuD), however, failed again. A new PCR was set up for this BioBrick.<br />
<br />
Some digests were set up. [http://partsregistry.org/Part:BBa_R0011 R0011]+[http://partsregistry.org/Part:BBa_B0033 B0033], [http://partsregistry.org/Part:BBa_K145013 K145013] and [http://partsregistry.org/Part:BBa_K145015 K145015] were cut with ''Eco''RI and ''Spe''I, and [http://partsregistry.org/Part:BBa_K145001 K145001]+[http://partsregistry.org/Part:BBa_B0015 B0015] was cut with ''Xba''I.<br />
<br />
We also electroporated a few of the longer ligations (step 2). We hope they will result in fluorescent colonies tomorrow. It concerns the following ligations: (J23116+B0032)+(C0040+B0015), (R0040+B0032)+(K145015+B0015), (I712074+J23032)+(C0012+B0015), (I712074+J23032)+(C0060+B0015) and (R0040+J23022)+(J23109+J23032).<br />
<br />
We also redid the PCR of the transduction. We will put these PCR samples on gel tomorrow.<br />
<br />
=== Dry Lab ===<br />
<br />
Worked on some applied bioethics and human practice<br />
<br />
==== Wiki ====<br />
<br />
The dropdown can now be docked by clicking the little square in the right top corner of the screen (the blueish with a bit of white in it), which is useful if you're browsing long pages. It doesn't work in IE, but that's an error that beyond my power... for real.<br />
<br />
Global styling has been updated, but can't be seen yet, or not anymore, as I accidentally reloaded the old global style, zoink! I'll change that tonight.<br />
<br />
Home will be updated tomorrow so it looks better.<br />
<br />
Future updates will include<br />
* modifying the edit box so you can have syntax highlighting<br />
* ...</div>BNathaliehttp://2008.igem.org/Team:KULeuven/3_September_2008Team:KULeuven/3 September 20082008-10-30T01:12:35Z<p>BNathalie: </p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
{{:Team:KULeuven/Tools/New_Day/Date_Retriever}}<br />
<br />
== Lab Work ==<br />
<br />
=== Wet Lab ===<br />
Once again, we tried to construct some of our new parts using PCR. It concerns the BioBricks K145014 (T7 polymerase with UmuD tag), K145150 (hybrid promoter) and K145013 (antisense LuxI).<br />
<br />
We continued yesterday's digests. [http://partsregistry.org/Part:BBa_C0040 C0040]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_C0060 C0060]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_C0012 C0012]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_K145015 K145015]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_K145151 K145151]+[http://partsregistry.org/Part:BBa_B0015 B0015] were cut with ''Eco''RI. They seemed alright.<br />
<br />
We started a few ligations. This is step 2 of the ligation process, so they are a bit longer: (J23116+B0032)+(C0040+B0015), (R0040+B0032)+(K145015+B0015), (I712074+J23032)+(C0012+B0015), (I712074+J23032)+(C0060+B0015) and (R0040+J23022)+(J23109+J23032).<br />
<br />
And finally, we also electroporated the ligations we made yesterday. Some of these ligations were electroporated into JM109 electrocompetent cells:<br />
* into TOP10 -> [http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_B0033 B0033], [http://partsregistry.org/Part:BBa_R0053 R0053]+[http://partsregistry.org/Part:BBa_P1052 P1052], [http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_J23066 J23066], [http://partsregistry.org/Part:BBa_B0014 B0014]+[http://partsregistry.org/Part:BBa_B0033 B0033], [http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_E0240 E0240], [http://partsregistry.org/Part:BBa_C0056 C0056]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_C0061 C0061]+[http://partsregistry.org/Part:BBa_B0015 B0015] - and of course pUC. <br />
* into JM109 -> [http://partsregistry.org/Part:BBa_C0056 C0056]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_C0061 C0061]+[http://partsregistry.org/Part:BBa_B0015 B0015] - and pUC.<br />
<br />
=== Dry Lab ===<br />
<br />
Ethics...<br />
(re)-writing sections on the wiki...<br />
LaTeX output tool for MATLAB...<br />
<br />
==== Modeling ====<br />
<br />
Working out how we can make MATLAB remember diffusion of HSL in the medium during the simulation. Also investigating the scale at which this diffusion happens in the simulation<br />
<br />
== Remarks ==<br />
<br />
Jonathan came to visit us. He's very interested in the iGEM competition and it was his first visit to a team. We had a very interesting conversation and it was a really inspiring visit.</div>BNathaliehttp://2008.igem.org/Team:KULeuven/2_September_2008Team:KULeuven/2 September 20082008-10-30T01:04:27Z<p>BNathalie: </p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
{{:Team:KULeuven/Tools/New_Day/Date_Retriever}}<br />
<br />
== Lab Work ==<br />
<br />
=== Wet Lab ===<br />
<br />
We tried to construct our parts [http://partsregistry.org/Part:BBa_K145150 K145150] (hybrid promoter) and [http://partsregistry.org/Part:BBa_K145014 K145014] (T7 polymerase with UmuD tag) using PCR. Both of them failed, but K145150 gave two bands. This is probably because we didn't purify it at first. We will do that tomorrow.<br />
<br />
We made electrocompetent cells of the JM109 stem.<br />
<br />
We did some more digests: <br />
* cut with ''Spe''I and ''Eco''RI -> [http://partsregistry.org/Part:BBa_R0040 R0040], [http://partsregistry.org/Part:BBa_B0014 B0014], [http://partsregistry.org/Part:BBa_R0053 R0053] and [http://partsregistry.org/Part:BBa_R1052 R1052](R1052 failed). <br />
* cut with ''Xba''I -> [http://partsregistry.org/Part:BBa_C0040 C0040]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_C0060 C0060]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_C0012 C0012]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_K145015 K145015]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_K145151 K145151]+[http://partsregistry.org/Part:BBa_B0015 B0015].<br />
<br />
We started a few ligations: [http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_B0033 B0033], [http://partsregistry.org/Part:BBa_R0053 R0053]+[http://partsregistry.org/Part:BBa_P1052 P1052], [http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_J23066 J23066], [http://partsregistry.org/Part:BBa_B0014 B0014]+[http://partsregistry.org/Part:BBa_B0033 B0033] and [http://partsregistry.org/Part:BBa_R0040 R0040]+[http://partsregistry.org/Part:BBa_E0240 E0240].<br />
<br />
We also tested the ligations of 31 August using PCR. Most of them were OK. The ligations that succeeded so far can be found on the schemes below.<br />
<br />
[[Image:2sept final.PNG|left|thumb|370px|Current Status (Final Scheme)]]<br />
[[Image:2sept parallel.PNG|center|thumb|387px|Current Status (Parallel Scheme)]]<br />
<br />
=== Dry Lab ===<br />
<br />
Ethics...<br />
<br />
==== Modeling ====<br />
<br />
Latex-tool...<br />
Multi-cell...<br />
Diffusion...<br />
Updating wiki...<br />
<br />
==== Wiki ====<br />
<br />
== Remarks ==</div>BNathaliehttp://2008.igem.org/File:Gl-01-9.jpgFile:Gl-01-9.jpg2008-10-30T00:51:50Z<p>BNathalie: </p>
<hr />
<div></div>BNathaliehttp://2008.igem.org/Team:KULeuven/Tools/HeaderTeam:KULeuven/Tools/Header2008-10-29T23:53:17Z<p>BNathalie: </p>
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<li id="abstract"><a title="See what's Dr.Coli all about" href="https://2008.igem.org/Team:KULeuven/Project"><strong>Project</strong> abstract</a></li><br />
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<a>The Team</a><br />
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<a href="https://2008.igem.org/Team:KULeuven/Team/LabsandGroups">Research Labs and Groups</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Team/Students">Students</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Team/Instructors">Instructors</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Team/Advisors">Advisors</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Team/Pictures">Pictures</a><br />
</div><br />
</li><br />
<li><br />
<a>The Project</a><br />
<div><br />
<a href="https://2008.igem.org/Team:KULeuven/Project">Summary</a><br />
<span><br />
<a>Components</a><br />
<div><br />
<a href="https://2008.igem.org/Team:KULeuven/Project/Input">Input</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Project/Output">Output</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Project/Filter">Filter</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Project/Inverter">InverTimer</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Project/Reset">Reset</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Project/CellDeath">Cell Death</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Project/Memory">Memory</a><br />
</div><br />
</span><br />
<a href="https://2008.igem.org/Team:KULeuven/Safety">Safety</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Evaluation">End Evaluation</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Literature">Literature</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Brainstorm">Brainstorm</a><br />
</div><br />
</li><br />
<li><br />
<a>Ethics</a><br />
<div><br />
<a href="https://2008.igem.org/Team:KULeuven/Ethics/Introduction">Introduction</a><br />
<span><br />
<a>Three A's</a><br />
<div><br />
<a href="https://2008.igem.org/Team:KULeuven/Ethics/Threeas/Defining">Defining the three A's</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Ethics/Threeas/Physics">Developments in modern physics</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Ethics/Threeas/Biology">Developments in biology</a><br />
</div><br />
</span><br />
<span><br />
<a>Concerns & Issues</a><br />
<div><br />
<a href="https://2008.igem.org/Team:KULeuven/Ethics/Concerns/Defining">Defining synthetic biology</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Ethics/Concerns/Framework">A framework</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Ethics/Concerns/Concerns">Concerns</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Ethics/Concerns/Issues">Issues</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Ethics/Concerns/Answers">The need for answers</a><br />
</div><br />
</span><br />
<span><br />
<a>Biological Robotics</a><br />
<div><br />
<a href="https://2008.igem.org/Team:KULeuven/Ethics/Robotics/KULeuven">KULeuven iGEM 2008 project</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Ethics/Robotics/Robotics">Biological robotics</a><br />
</div><br />
</span><br />
<a href="https://2008.igem.org/Team:KULeuven/Ethics/Afterword">Afterword</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Ethics/References">References</a><br />
</div><br />
</li><br />
<li><br />
<a>Submitted Parts</a><br />
<div><br />
<a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=KULeuven">Sandbox</a><br />
</div><br />
</li><br />
<li><br />
<a>Modeling</a><br />
<div><br />
<a href="https://2008.igem.org/Team:KULeuven/Model/Overview">Overview</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Model/KineticConstants">Kinetic Constants</a><br />
<span><br />
<a>Components</a><br />
<div><br />
<a href="https://2008.igem.org/Team:KULeuven/Model/Output">Output</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Model/Filter">Filter</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Model/Inverter">InverTimer</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Model/Reset">Reset</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Model/CellDeath">Cell Death</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Model/Memory">Memory</a><br />
</div><br />
</span><br />
<a href="https://2008.igem.org/Team:KULeuven/Model/FullModel">Full Model</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Model/Sensitivity">Sensitivity Analysis</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Model/MultiCell">Multi-cell Model</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Model/Diffusion">Diffusion</a><br />
</div><br />
</li><br />
<li><br />
<a>Data Analysis</a><br />
<div><br />
<a href="https://2008.igem.org/Team:KULeuven/Data/Overview">Overview</a><br />
<span><br />
<a>New Parts</a><br />
<div><br />
<a href="https://2008.igem.org/Team:KULeuven/Data/GFP">GFP (LVA-tag)</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Data/Other_Parts">Other</a><br />
</div><br />
</span><br />
<span><br />
<a>Components</a><br />
<div><br />
<a href="https://2008.igem.org/Team:KULeuven/Data/Input">Input</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Data/Output">Output</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Data/Other_Components">Other</a><br />
</div><br />
</span><br />
</div><br />
</li><br />
<li><br />
<a>Software</a><br />
<div><br />
<a href="https://2008.igem.org/Team:KULeuven/Software/MultiCell">Multi-cell Toolbox</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Software/Simbiology2LaTeX">Simbiology2LaTeX Toolbox</a><br />
</div><br />
</li><br />
<li><br />
<a>Notebook</a><br />
<div><br />
<a href="https://2008.igem.org/Team:KULeuven/Protocols">Protocols</a><br />
<span><br />
<a>Reports</a><br />
<div><br />
<a href="https://2008.igem.org/Team:KULeuven/Meeting Reports">Daily</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Weekly Meetings">Weekly</a><br />
</div><br />
</span><br />
<span><br />
<a>Lab Data</a><br />
<div><br />
<a href="https://2008.igem.org/Team:KULeuven/Freezer">Freezer</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Primers">Primers</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Ligation">Ligation</a><br />
</div><br />
</span><br />
<a href="https://2008.igem.org/Team:KULeuven/Tools">Tools</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Press">Press</a><br />
<a href="https://2008.igem.org/Team:KULeuven/Acknowledgments">Acknowledgments</a><br />
</div><br />
</li><br />
<br />
</ul><br />
<br />
</div><br />
<!-- END main navigation --><br />
<br />
</div><br />
</html></div>BNathaliehttp://2008.igem.org/Team:KULeuven/AcknowledgmentsTeam:KULeuven/Acknowledgments2008-10-29T23:47:46Z<p>BNathalie: </p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
== BioBricks ==<br />
<br />
* [http://parts2.mit.edu/wiki/index.php/University_of_California_Berkeley_2006 Berkeley 2006 team] for their fantastic work on the RiboRegulators<br />
<br />
== Wiki ==<br />
<br />
* [https://2007.igem.org/Paris Paris 2007 team] for their inspirational lab templates, to be found on our [[Team:KULeuven/Tools|tools]] page<br />
* [[Team:NYMU-Taipei|NYMU-Taipei 2008 team]] for their inspirational image map, which we adapted for the team picture on our [[Team:KULeuven|homepage]]<br />
<br />
== Sponsors ==<br />
{|width="90%"<br />
|[[image:Kul.jpg|center|200px]]<br />
|[[image:bioscenter.jpg|right|250px]]<br />
|[[image:mathworks.jpg|left|200px]]<br />
|}</div>BNathaliehttp://2008.igem.org/File:Mathworks.jpgFile:Mathworks.jpg2008-10-29T23:44:02Z<p>BNathalie: </p>
<hr />
<div></div>BNathaliehttp://2008.igem.org/File:Bioscenter.jpgFile:Bioscenter.jpg2008-10-29T23:43:43Z<p>BNathalie: </p>
<hr />
<div></div>BNathaliehttp://2008.igem.org/File:Kul.jpgFile:Kul.jpg2008-10-29T23:43:24Z<p>BNathalie: </p>
<hr />
<div></div>BNathaliehttp://2008.igem.org/Team:KULeuven/Data/Other_PartsTeam:KULeuven/Data/Other Parts2008-10-29T23:28:12Z<p>BNathalie: </p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Scripting}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
[[Image:logo-other(1).jpg|120px|right]]<br />
<br />
== Introduction ==<br />
<br />
As Dr.Coli is a very extensive project, there was not sufficient time to test all the new parts that we created. This page contains a summary of how our new basic parts can be tested (the construct that have to be made and the actual testing protocol). It will also give an overview of the results we had so far. We worked as much in parallel as possible, building different parts and subsystems at the same time. This way we could still have some nice results, even when some of the parts didn't succeed. All the new parts introduced in our project were properly built. We leave the actual testing of these parts to the next year's teams, our advisors or you :-).<br />
<br />
== Antisense LuxI ==<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K145013 Parts Registry:K145013]<br />
<br />
=== Construct & protocol ===<br />
<br />
Antisense LuxI (asLuxI) is an RNA molecule that can bind to the mRNA of ''luxI'', thus repressing the translation of the LuxI enzyme. Testing this new part would not be very easy. One would need two rather complex systems :<br />
<br />
{|style="background:#ffffff; text-align:center; width:90%" <br />
|'''System 1:'''||[[Image:K145201b.JPG|137px|center]]||+||[[Image:test-aslux(1).JPG|140px|center]]||+||[[Image:test-aslux(2).JPG|140px|center]]||+||[[Image:test-aslux(3).JPG|140px|center]]||||<br />
|-<br />
|'''System 2:'''||[[Image:K145201b.JPG|137px|center]]||+||[[Image:test-aslux(1).JPG|140px|center]]||+||[[Image:test-aslux(2).JPG|140px|center]]||+||[[Image:test-aslux(3).JPG|140px|center]]||+||[[Image:test-aslux(4).JPG|130px|center]]<br />
|}<br />
<br><br />
In system 1 LuxR is produced constitutively. On its own LuxR will not do much, but when it is bound to HSL it can activate the transcription of ''gfp'' from the ''lux'' promoter (R0062). HSL is a molecule that can be generated by LuxI, and the production of LuxI depends on the presence or absence of aTc. So, when no aTc (to relieve TetR repression) is present, there will be no LuxI and therefore no HSL, and no active LuxR. This means no production of GFP. When aTc is added to the system, LuxI will be produced and so will HSL. HSL can then bind to LuxR and start the transcription of GFP (fluorescence should become detectable).<br />
<br />
System 2 works very much in the same way, but now we also have the constitutive production of antisense LuxI. This means that, when aTc is added to the system, transcription of ''luxI'' will occur. However, the protein LuxI should not be produced in high amounts because ''asLuxI'' will bind the mRNA of ''luxI''. No LuxI means no HSL, and no active LuxR, so no GFP. In system 2 the production of GFP will never occur, with or without aTc.<br />
<br />
To test this, one should build these two systems and transform them into ''E. coli'' cells. After that you can prepare liquid cultures of the 2 systems. Then you should add aTc (100 ng/ml) and follow the fluorescence of the liquid cultures in time (e.g. with FACS). After incubation and if all goes well, you should see highly fluorescent cells with system 1, while the fluorescence of system 2 should remain low.<br />
<br />
=== Results ===<br />
<br />
Antisense LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K145013 K145013]) was constructed with PCR. The template was the plasmid containing the ''luxI'' BioBrick ([http://partsregistry.org/wiki/index.php?title=Part:BBa_C0061 C0061]). We used the following primers :<br />
<div style="margin-left:90px;"> Forward Primer: CATCAGGAAT TCGCGGCCGC TTCTAGTTAT TAAGCTACTA AAGCGTAGTT TTC</div><br />
<div style="margin-left:90px;"> Forward Primer: CTGCAGCGGC CGCTACTAGT AATGACTATA ATGATAAAAA AATCGG</div><br />
<br />
This PCR succeeded very well and we then cut the PCR product with ''Eco''RI and ''Spe''I. This digest was consecutively ligated into a pSB1A2 plasmid with T4 DNA ligase. This plasmid was then electroporated into TOP10 cells and these cells were grown on agar plates containing ampicillin. Everything went well and sequencing showed that part K145013 was built correctly !<br />
<br />
== T7 RNA polymerase (with and without UmuD) ==<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K145014 Parts Registry:K145014] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K145001 Parts Registry:K145001]<br />
<br />
=== Construct & protocol ===<br />
<br />
T7 RNA polymerase is an enzyme that will start transcription from a T7 promoter. We use this part in our filter and there it is necessary that this enzyme is degraded quickly. Therefore we also constructed a T7 polymerase with an UmuD tag. This tag will render T7 RNA polymerase susceptible to degradation by certain proteases. To test whether this tag really makes T7 polymerase degrade faster, one should build the following constructs:<br />
<br />
{|style="background:#ffffff; text-align:center; width:55%" <br />
|'''Construct 1 (no tag):'''||[[Image:K145201b.JPG|center]]||+||[[Image:test-T7(1).JPG|center]]<br />
|-<br />
|'''Construct 2 (UmuD tag):'''||[[Image:K145201b.JPG|center]]||+||[[Image:test-T7(2).JPG|center]]<br />
|}<br />
<br><br />
In these two constructs, TetR is constitutively produced and this will repress the production of T7 RNA polymerase from the ''tet'' promoter (R0040). Adding aTc will render TetR inactive and T7 RNA polymerase will be produced. So, in order to test the degradation of the enzymes, you should grow liquid cultures containing aTc of the cells with construct 1 or construct 2. These cells will start to produce T7 RNA polymerase or T7 RNA polymerase with UmuD. When these cells have been grown overnight, you can transfer them to another medium that does not contain aTc. The production of T7 polymerase will then stop and you can now measure the degradation rate of the T7 RNA polymerases. To do this, you should harvest some of the cells at several time intervals. Then you should isolate the proteins from the cells and determine the concentration of T7 RNA polymerase using anti-T7 antibodies. The tag works if the concentration of T7 RNA polymerase with UmuD tag decreases much faster in time than the concentration of normal T7 RNA polymerase.<br />
<br />
=== Results ===<br />
<br />
We constructed both T7 RNA polymerases with a PCR reaction. To built the T7 RNA polymerase without the tag ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K145001 K145001]), we did a PCR with pfx polymerase on the plasmid of [http://partsregistry.org/wiki/index.php?title=Part:BBa_I712022 I712022] as template and with the following primers:<br />
<div style="margin-left:90px;">Forward primer: ATGAATTCGC GGCCGCTTCT AGATGAACAC GATTAACATC GC</div><br />
<div style="margin-left:90px;">Reverse primer: CTGCAGCGGC CGCTACTAGT ATTATTACGC GAACGCGAAG TCCG</div><br />
<br />
<br />
To attach an UmuD tag to the T7 RNA polymerase ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K145014 K145014]) we did a two-step PCR. The first PCR was done with I712022 as a template and the following primers:<br />
<div style="margin-left:90px;">Forward primer: CCGCTATTTA GCGATCTTGT TCAGTGTGGC TTTCCTTCAC CGATGAACAC GATTAACATC GC</div><br />
<div style="margin-left:90px;">Reverse primer: CTGCAGCGGC CGCTACTAGT ATTATTACGC GAACGCGAAG TCCG</div><br />
<br />
<br />
The second step of the PCR was performed on the unpurified PCR product of step one and with these two primers:<br />
<div style="margin-left:90px;">Forward primer: ATGAATTCGC GGCCGCTTCT AGATGTTGTT TATCAAGCCT GCGGATCTCC GCGAAATTGT GACTTTTCCG</div> <div style="margin-left:185px;">CTATTTAGCG ATCTTGTTCA G</div><br />
<div style="margin-left:90px;">Reverse primer: CTGCAGCGGC CGCTACTAGT ATTATTACGC GAACGCGAAG TCCG</div><br />
<br />
<br />
It took very long, but eventually the two PCRs succeeded. These PCR products were then cut with ''Eco''RI and ''Xba''I, and these digests were ligated into pSB1A2 plasmids. These plasmids were electroporated into TOP10 cells and the obtained colonies were miniprepped. Sequencing showed that both PCRs succeeded very well and that we made the correct constructs ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K145001 K145001], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K145014 K145014]).<br />
<br />
The first part of the test constructs (J23116+B0034+C0040+B0015) is actually our input and this was properly built, see [https://2008.igem.org/Team:KULeuven/Data/Input here]. We also tried to build the second part (R0040+B0032+T7polymerase+B0015), but various attempts to ligate the T7 polymerase genes to B0015 failed.<br />
<br />
== Hybrid promoter ==<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K145150 Parts Registry:K145150]<br />
<br />
=== Construct & protocol ===<br />
<br />
The hybrid promoter (HPr) is a part that was completely designed by ourselves and it is used in our cell death mechanism. This is a promoter that can be regulated in two ways. On the one hand it can be activated by a complex of LuxR-HSL and on the other hand it can be repressed by cII P22. In order to test if this promoter works properly, we designed two seperate tests.<br />
<br />
[[Image:K145267b.JPG|center]]<br />
The first test is designed to see if the LuxR-HSL complex can activate transcription from this promoter. We would need the construct that is shown above. This construct will constitutively produce LuxR. LuxR on its own cannot activate transcription from the hybrid promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K145150 K145150]) and so production of GFP will be only basal. However, when we add HSL, the transcription of ''gfp'' should become activated. So, if you want to test whether the hybrid promoter can be activated, you would have to make a liquid culture of the cells with the above construct. Then you can add HSL to the liquid culture and start measuring the fluorescence of the cells at different time intervals (e.g. with FACS). If all goes well, the fluorescence should increase upon addition of HSL.<br />
<br />
[[Image:K145266b.JPG|center]]<br />
The second test is designed to see if cII P22 can repress transcription from the hybrid promoter, even in the presence of a LuxR-HSL complex. Therefore we should build the construct shown above. In this construct we have constitutive production of TetR, which will repress the production of cII P22. When aTc is added to the cells, TetR is inactivated and cII P22 is produced. This will then act as a repressor for the hybrid promoter, thus completely blocking the expression of GFP. We also have constitutive production of LuxR, but this shouldn’t do anything. It should only activate the expression of GFP when HSL is present and cII P22 is absent. So, if you want to test the hybrid promoter, you should prepare some liquid cultures of cells containing this construct and add various amounts of aTc and/or HSL. Then you can see what the effect is of these molecules by measuring the fluorescence at different time intervals (e.g. with FACS). If the hybrid promoter works properly, the results should look like this:<br />
{| style="background:#99CCFF; color:black; width:60%; text-align:center;" border="1" cellpadding="2" cellspacing="0"<br />
|Molecules added||Fluorescence||Molecules added||Fluorescence<br />
|- style="background:#ffffff; color:black; text-align:center;"<br />
|nothing||no||HSL + aTc||no<br />
|- style="background:#ffffff; color:black; text-align:center;"<br />
|only HSL||yes||only aTc||no<br />
|}<br />
<br />
=== Results ===<br />
<br />
The hybrid promoter itself was built by an end-filling reaction using the Klenow fragment (polymerase). The primers that were used in this end-filling reaction and that make up the promoter were the following:<br />
<div style="margin-left:90px;">Forward primer: CATCATGAAT TCGCGGCCGC TTCTAGACCT GTAGGATCGT ACAGGTTTAC TAAAGATTCC TTTAG</div><br />
<div style="margin-left:90px;">Reverse primer: TACTAGTAGC GGCCGCTGCA GGAAATTAAA GAACACTTAA ATTATAAACT AAAGGAATCT TTAG</div><br />
<br />
When this was finished, we digested it with ''Eco''RI and ''Spe''I and ligated it into a pSB1A2 plasmid. This plasmid was later on electroporated into ''E.coli'' cells and then miniprepped. We also tried to make the construct for the first test and we actually performed this test, but it failed. Sequencing then showed that our construct didn’t contain all the desired parts. We also tried to sequence the hybrid promoter, but this failed twice.<br />
<br />
== ccdB ==<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K145151 Parts Registry:K145151]<br />
<br />
=== Construct & protocol ===<br />
<br />
In our project we want Dr.Coli to eliminate himself when he is not needed anymore. We do this because we don’t want useless bacteria in our gut or the environment. One way to achieve this is by using the protein CcdB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K145151 K145151]). This protein is toxic to ''E.coli'' cells (without CcdA) and therefore very suitable. Constructing and testing this part would be relatively simple. To test whether our CcdB can cause cell death, we should construct the following BioBrick:<br />
{|style="background:#ffffff; text-align:center; width:40%" <br />
|[[Image:K145201b.JPG|center]]||+||[[Image:test-ccdB.JPG|center]]<br />
|}<br />
<br><br />
In this construct the production of CcdB is inhibited by the constitutive production of the Tet repressor. Adding aTc however, will inactivate this repressor and CcdB will be produced, causing the cells to die. To test the function of CcdB you should prepare three types of agar plates: <br />
# one plate should be inoculated with Top10 cells containing the construct and aTc should be present<br />
# another plate should be inoculated with the same cells containing the same construct, but aTc should not be present<br />
# yet another plate should be inoculated with empty Top10 cells (no plasmid) and aTc should be present<br />
The results of this test should be that the first plate (CcdB induced by aTc) contains considerably fewer colonies – preferably none – than the other to plates. If you want to be sure that the lack of colonies is not just a coincidence, you should repeat this test a few times. When you always get the same results (no colonies on the first plate), then this CcdB construct works.<br />
<br />
=== Results ===<br />
<br />
The ''ccdB'' gene was obtained by PCR on the plasmid of BioBRick [http://partsregistry.org/wiki/index.php?title=Part:BBa_P1010 P1010] as a template with the following primers:<br />
<div style="margin-left:90px;">Forward primer: CATCATGAAT TCGCGGCCGC TTCTAGATGC AGTTTAAGGT TTACACC</div><br />
<div style="margin-left:90px;">Reverse primer: CTGCAGCGGC CGCTACTAGT ATTATATTCC CCAGAACATC AGG</div><br />
<br />
<br />
This PCR didn’t cause any problems and the PCR product was cut with ''Eco''RI and ''Spe''I. This digest was then ligated into pSB1A2 cells and subsequently electroporated into Top10 cells and miniprepped. Sequencing showed that CcdB was constructed correctly. We also started to build the test construct, but unfortunately we couldn’t finish it.</div>BNathaliehttp://2008.igem.org/Team:KULeuven/Model/MultiCellTeam:KULeuven/Model/MultiCell2008-10-29T23:26:19Z<p>BNathalie: </p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Scripting}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
== Multi-cell Modeling ==<br />
<br />
[[Image:Multicell icon.PNG|right|300px]]<br />
<br />
The output of our InverTimer, the LuxI protein, causes an accumulating amount of HSL molecules, which are in fact signaling molecules. This means that they are used by cells to communicate with each other by diffusing in the environment. <br />
<br />
At this point we have only simulated 1 Dr. Coli @ work. It might be a good idea to take this model to a higher level and simulate the behaviour of a colony of cells. At a later stage we can add a diffusive environment (medium), in which the signaling molecules can travel from one cell to a neighboring cell, and see what happens...<br />
<br />
=== Full Model ===<br />
<br />
A small example leads to a simple conclusion: this analysis is unnecessary. Even when there is possible diffusion of HSL molecules to the environment, it won't happen because of the exces amount of LuxR molecules. LuxR will immediately bind the available HSL molecules (very high association rate) and no HSL will diffuse.<br />
<br />
[[Image:multicell.png|center|400px]]<br />
<br />
<br />
=== Full Model - Part 2 ===<br />
<br />
Because of the auto-inducible LuxR promotor there will certainly be diffusion into the medium (see [https://2008.igem.org/Team:KULeuven/Model/FullModel#Full_Model_-_Part_2 Model - Part2]): a deeper research needs to be done! Therefor we wrote a Matlab-script which is capable of interacting with the Simbiology Toolbox. This enables us to replicate cells in silico: we can simulate an increasing population of cells. A pretty visualisation is given below: each pop-up of a bar simulates the sudden presence of a new cell caused by the division of one of the available cells (starting population exists of 4 cells). This sudden pop-up effect is because we only simulate cell multiplication and not the growth of the new born cells: they are immediately at full size with about the same concentrations of the mother cell.<br />
<br />
[[Image:Sim1.png|center|600px]]<br />
<br />
Second, we also implemented some functions to be able to simulate the whole diagram with events and some other features which are present in the Simbiology Toolbox. At this point we are able to run simulations with a growing (Dr. Coli) cell population while they are susceptible to external signals (TetR-input).<br />
<br />
<html><br />
<div class="center"><br />
<div class="noborder" style="overflow: auto; width: 800px; height: 420px;"><br />
<div class="noborder" style="width: 1250px;"> <br />
<img src="https://static.igem.org/mediawiki/2008/9/92/Multicell_HSL1.png" style="float: left; width: 600px; height: 400px; margin: 0 5px;" /><br />
<img src="https://static.igem.org/mediawiki/2008/0/0f/Multicell_ccdB1.png" style="float: left; width: 600px; height: 400px; margin: 0 5px;" /><br />
</div></div></div></html></div>BNathaliehttp://2008.igem.org/Team:KULeuven/Model/MemoryTeam:KULeuven/Model/Memory2008-10-29T23:24:40Z<p>BNathalie: </p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Scripting}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
[[Image:pictogram_memory.png|120px|right]]<br />
<br />
== Memory ==<br />
<br />
=== Position in the system ===<br />
<br />
This system must activate the cell death system after one light pulse. As long as there is no light, there is no P2ogr, no CIIP22 and a lot of antimRNA_LuxI. The antimRNA_LuxI blocks the cell death system. When light is turned on OmpF increases. This causes P2ogr and CIIP22 to increase and antimRNA_LuxI to decrease. This activates the system. When light is turned off, the P2ogr concentration is large enough to maintain itself. This way antimRNA doesn't increase. The system stays activated.<br />
<br />
=== Describing the system ===<br />
<br />
[[Image:Memory_BioBrick.jpg|center]]<br />
<br />
==== ODE's ====<br />
<br />
==== Parameters ====<br />
<br />
{| width=80% style="border: 1px solid #003E81; background-color: #EEFFFF;"<br />
|+ ''Parameter values (Memory)''<br />
! width=15% | Name<br />
! width=15% | Value<br />
! width=40% | Comments<br />
! width=10% | Reference<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Degradation Rates<br />
|-<br />
| d<sub>P2ogr</sub><br />
| 0.002265 s<sup>-1</sup><br />
|<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [1<html>]</html>]<br />
|-<br />
| d<sub>RNA_P2ogr</sub><br />
| 0.002265 s<sup>-1</sup><br />
| <br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [2<html>]</html>]<br />
|-<br />
| d<sub>P22CII</sub><br />
| 0.002311 s<sup>-1</sup><br />
| This value is too low. The correct value is used in the final model.<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [3<html>]</html>]<br />
|-<br />
| d<sub>RNA_P22CII</sub><br />
| 0,0022651 s<sup>-1</sup><br />
| <br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [2<html>]</html>]<br />
|-<br />
| d<sub>antimRNA_luxI</sub> <br />
| 0.0045303 s<sup>-1</sup><br />
| estimate: because this RNA isn't translated, it degrades faster<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [2<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Transcription Rates<br />
|-<br />
| k<sub>P2ogr</sub><br />
| 0.0125 s<sup>-1</sup><br />
| estimate<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [4<html>]</html>]<br />
|-<br />
| k<sub>P22CII</sub><br />
| 0.0125 s<sup>-1</sup><br />
| estimate<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [4<html>]</html>]<br />
|-<br />
| k<sub>AntimRNA_LuxI</sub><br />
| 0.0094 s<sup>-1</sup><br />
| estimate<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [5<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Dissociation Constants<br />
|-<br />
| K<sub>P2ogr</sub><br />
| 4.2156<br />
| Used in two reactions for activator control at the transcription of P2ogr mRNA and CIIP22 mRNA<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [6<html>]</html>]<br />
|-<br />
| K<sub>R0053_P22CII</sub><br />
| 0.1099<br />
| <br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [3<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Hill Cooperativity <br />
|-<br />
| n<sub></sub><br />
| 2<br />
| Used for all reactions throughout the memory submodel using Hill kinetics<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [1<html>]</html>]<br />
|-<br />
|}<br />
<br />
=== Models ===<br />
<br />
==== CellDesigner ([https://static.igem.org/mediawiki/2008/4/49/Memory_CellDesigner.zip SBML file])====<br />
<br />
[[Image:Memory.png|600px|center|Memory]]<br />
<br />
==== Matlab ====<br />
<br />
=== Problem ===<br />
<br />
The OmpF promoter is not ideal. When there is no light the transcription rate is still 0.00005 s<sup>-1</sup>. This means that P2ogr will slowly build up, activating the system. In this case the memory is in 0-state when the stationary state isn't reached yet. The 1-state is the stationary state. So the system automatically ends up in state 1 after some time (300s). This can be seen in the figure below.<br />
[[Image:mem_no_act.png|600px|center|mem_no_act]]<br />
::: '''Figure: CIIP22(purple), P2ogr(green), AntimRNA(pink).<br />
The system can only stay in 0-state for 300 sec. This makes it completely useless.<br />
<br />
=== Alternative ===<br />
In the previous system the 0-state isn't actively maintained. It's just 'not stationary state'. So we need to search mathematical system that has 2 stationary states. A possible solution is given below.<br />
<br />
[[Image:Alt.png|500px|center|alt]]<br />
::::'''Figure: Part representation of alternative system'''<br />
<br />
When this system starts Rep build up because Rep represses the Act promoter better then Act represses the Rep promoter. The Rep concentration stays high and the Act stays low. This is the 0-state. When there is light, the OmpF promoter is activated and the Act concentration is increased. This represses Rep promoter. The Rep concentration decreases and the Act promoter is activated. The Act concentration keeps increasing. When the light pulse ends the Act concentration is high enough to repress the Rep promoter, Act concentration stays high and Rep concentration stays low. This is the 1-state.<br />
<br />
CellDesigner gives the following simulation when OmpF transcription rate changes from 0.0001 to 0.01 at t=6000 sec for 2000 sec.<br />
[[Image:alt_CDplot.png|600px|center|alt_CDplot]]<br />
:::'''Figure: Celldesigner simulation of the alterative system. Act(grey), Rep(yellow) '''<br />
The OmpF peak causes the Act concentration to rise and the Rep to decrease. The high Act concentration keeps the Rep concentration low. This causes the Act concentration to stay high.<br />
<br />
=== References ===<br />
<br />
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=utf-8"/><br />
<title>Bibliography</title><br />
</head><br />
<body><br />
<table style="border-collapse:collapse;line-height:1.1em;"><br />
<tr style="vertical-align:top;"><td>[1]</td><td style="padding-left:4pt;">“ETHZ/Parameters - IGEM07”; https://2007.igem.org/ETHZ/Parameters.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[2]</td><td style="padding-left:4pt;">J.A. Bernstein et al., “Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays,” <span style="font-style:italic;">Proceedings of the National Academy of Sciences of the United States of America</span>, vol. 99, Jul. 2002, pp. 9697–9702. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1073/pnas.112318199&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Global%20analysis%20of%20mRNA%20decay%20and%20abundance%20in%20Escherichia%20coli%20at%20single-gene%20resolution%20using%20two-color%20fluorescent%20DNA%20microarrays&amp;rft.jtitle=Proceedings%20of%20the%20National%20Academy%20of%20Sciences%20of%20the%20United%20States%20of%20America&amp;rft.stitle=Proc%20Natl%20Acad%20Sci%20U%20S%20A.%20&amp;rft.volume=99&amp;rft.issue=15&amp;rft.aufirst=Jonathan%20A.&amp;rft.aulast=Bernstein&amp;rft.au=Jonathan%20A.%20Bernstein&amp;rft.au=Arkady%20B.%20Khodursky&amp;rft.au=Pei-Hsun%20Lin&amp;rft.au=Sue%20Lin-Chao&amp;rft.au=Stanley%20N.%20Cohen&amp;rft.date=2002-07-23&amp;rft.pages=9697%E2%80%939702"></span></td></tr><br />
<br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[3]</td><td style="padding-left:4pt;">J. De Anda, A. Poteete, and R. Sauer, “P22 c2 repressor. Domain structure and function,” <span style="font-style:italic;">J. Biol. Chem.</span>, vol. 258, Sep. 1983, pp. 10536-10542. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=P22%20c2%20repressor.%20Domain%20structure%20and%20function&amp;rft.jtitle=J.%20Biol.%20Chem.&amp;rft.volume=258&amp;rft.issue=17&amp;rft.aufirst=J&amp;rft.aulast=De%20Anda&amp;rft.au=J%20De%20Anda&amp;rft.au=AR%20Poteete&amp;rft.au=RT%20Sauer&amp;rft.date=1983-09-10&amp;rft.pages=10536-10542"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[4]</td><td style="padding-left:4pt;">“Part:BBa I746364 - partsregistry.org”; http://partsregistry.org/Part:BBa_I746364.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[5]</td><td style="padding-left:4pt;">“Part:BBa R0053 - partsregistry.org”; http://partsregistry.org/Part:BBa_R0053.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[6]</td><td style="padding-left:4pt;">D. Kornitzer, S. Altuvia, and A.B. Oppenheim, “The activity of the CIII regulator of lambdoid bacteriophages resides within a 24-amino acid protein domain.,” <span style="font-style:italic;">Proceedings of the National Academy of Sciences of the United States of America</span>, vol. 88, Jun. 1991, pp. 5217–5221. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=The%20activity%20of%20the%20CIII%20regulator%20of%20lambdoid%20bacteriophages%20resides%20within%20a%2024-amino%20acid%20protein%20domain.&amp;rft.jtitle=Proceedings%20of%20the%20National%20Academy%20of%20Sciences%20of%20the%20United%20States%20of%20America&amp;rft.stitle=Proc%20Natl%20Acad%20Sci%20U%20S%20A.%20&amp;rft.volume=88&amp;rft.issue=12&amp;rft.aufirst=D.&amp;rft.aulast=Kornitzer&amp;rft.au=D.%20Kornitzer&amp;rft.au=S.%20Altuvia&amp;rft.au=A%20B%20Oppenheim&amp;rft.date=1991-06-15&amp;rft.pages=5217%E2%80%935221"></span></td></tr><br />
<br />
<tr><td colspan="2">&nbsp;</td></tr><br />
</table></body><br />
</html><br />
<br />
==Memory - episode 2==<br />
<br />
=== Position in the system ===<br />
<br />
The Memory must keep the [https://2008.igem.org/Team:KULeuven/Model/Cell_Death Cell Death] inactivated untill a decent input signal has been received. When there is no input signal, c2 P22 will take control and repress cI 434 and [https://2008.igem.org/Team:KULeuven/Model/Cell_Death Cell Death]. This is the OFF state in which the memory can remain indefinitely unless an input signal emerges. This causes production of cI 434 (without LVA tag) whih will repress c2 P22 production, allowing cI 434 production to start from the c2 P22 repressible promoter. cI 434 will also start repressing the antisense LuxI production, enabling the [https://2008.igem.org/Team:KULeuven/Model/Inverter InverTimer] to do its work. After an input signal of about 1000 seconds, enough of the c2 P22 has disappeared in order to make the cI 434 production (LVA) self-sufficient. At this point, the memory has reached the ON state in which it will remain. For a more elaborate description of the Memory's biomolecular workings, please see the [https://2008.igem.org/Team:KULeuven/Project/Memory Project/Memory] page.<br />
<br />
=== Describing the system ===<br />
<br />
[[Image:Alt2.png|center]]<br />
<br />
This is the same figure as the alternative, but this time filled in for the new system. It conventiently shows that the system is based upon the alternative suggested above while laying out the workings and concretisations in the form of the different BioBricks implemented. Rep is now the c2 P22 repressor while Act is represented by cI 434.<br />
<br />
[[Image:New_Mem_symbols.PNG|center]]<br />
[[Image:New_Mem_symbols_output.PNG|center]]<br />
<br />
<br><br />
<br />
The output of this system is antisense RNA production that represses the LuxI translation. c2 P22 has also got a function in repressing [https://2008.igem.org/Team:KULeuven/Model/Cell_Death Cell Death]<br />
<br />
====ODE's====<br />
<html><br />
<body><br />
<p><br />
<a href="https://static.igem.org/mediawiki/2008/4/4c/MemoryODE.pdf"><br />
<img border="0" src="https://2008.igem.org/wiki/skins/common/images/icons/fileicon-pdf.png" width="65" height="60"><br />
</a><br />
</p><br />
</body><br />
</html><br />
<br />
==== Parameters ====<br />
{| width=80% style="border: 1px solid #003E81; background-color: #EEFFFF;"<br />
|+ ''Parameter values (New Memory)''<br />
! width=15% | Name<br />
! width=15% | Value<br />
! width=40% | Comments<br />
! width=10% | Reference<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Degradation Rates<br />
|-<br />
| d<sub>cIIP22</sub><br />
| d<sub>LVA</sub> = 2.814E-4 s<sup>-1</sup><br />
| LVA-tag reduces lifetime to 40 minutes<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [5<html>]</html>]<br />
|-<br />
| d<sub>mRNA_cIIP22</sub><br />
| 0.0023104906 s<sup>-1</sup><br />
| <br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [3<html>]</html>]<br />
|-<br />
| d<sub>cI434_LVA</sub><br />
| d<sub>LVA</sub> = 2.814E-4 s<sup>-1</sup><br />
| LVA-tag reduces lifetime to 40 minutes<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [5<html>]</html>]<br />
|-<br />
| d<sub>mRNA_cI434_LVA</sub><br />
| 0.0023104906 s<sup>-1</sup><br />
| <br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [3<html>]</html>]<br />
|-<br />
| d<sub>cI434</sub><br />
| 9.627044174E-5 s<sup>-1</sup><br />
| no LVA tag, so longer lifetime (t<sub>1/2</sub> = 2h)<br />
| <br />
|-<br />
| d<sub>mRNA_cI434</sub><br />
| 0.0023104906 s<sup>-1</sup><br />
| <br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [3<html>]</html>]<br />
|-<br />
| d<sub>asRNA luxI</sub> <br />
| 0.00462 s<sup>-1</sup><br />
| estimate: because this RNA isn't translated, it degrades faster <br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [3<html>]</html>]<br />
|-<br />
| d<sub>LuxI asRNA complex</sub><br />
| 0.00462 s<sup>-1</sup><br />
| estimate: because this RNA isn't translated, it degrades faster <br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [3<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | c2 P22 transcription (cI 434 regulated)<br />
|-<br />
| k<sub>transcr</sub><br />
| 0.0125 s<sup>-1</sup><br />
| estimate of maximal transcription rate, strong promoter<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [10<html>]</html>]<br />
|-<br />
| K<sub>m</sub> (cI 434)<br />
| 0.8708<br />
| Recalculated to remove [M] dimension from K<sub>m</sub>=2E-9 [M]<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [12<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | cI 434 transcription (c2 P22 regulated)<br />
|-<br />
| k<sub>transcr</sub><br />
| 0.0040 s<sup>-1</sup><br />
| estimate of maximal transcription rate, weak-medium promoter<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [11<html>]</html>]<br />
|-<br />
| K<sub>m</sub> (c2 P22)<br />
| 0.1099<br />
| Recalculated to remove [M]<sup>2</sup> dimension from K<sub>app</sub>=6.8E-20[M]<sup>2</sup><br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [6<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | TetR regulated promoter<br />
|-<br />
| TetR_var_transcr_rate<br />
| p(TetR) dependent <br />
| (RiboKey) between 5E-5 and 0.0125 s<sup>-1</sup> ~ [aTc]<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [4<html>]</html> [9<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Translation rates<br />
|-<br />
| k<sub>transl_cI434</sub><br />
| 0.0055555 s<sup>-1</sup><br />
| translation rate for B0033 RBS (0.01 relative efficiency)<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [8<html>]</html>]<br />
|-<br />
| k<sub>transl_cI434_lva</sub><br />
| 0.0388889 s<sup>-1</sup><br />
| translation rate for B0031 RBS (0.07 relative efficiency)<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [7<html>]</html>]<br />
|-<br />
| k<sub>transl_cIIP22</sub><br />
| 0.0055555 s<sup>-1</sup><br />
| translation rate for B0033 RBS (0.01 relative efficiency)<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [8<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Hill Cooperativity <br />
|-<br />
| n<sub></sub><br />
| 2<br />
| Used for all reactions throughout the memory submodel using Hill kinetics<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [2<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Antisense LuxI<br />
|-<br />
| k_complex3<br />
| 0.00237 s<sup>-1</sup><br />
| rate constant for formation of asRNA - LuxI mRNA duplex<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [1<html>]</html>]<br />
|-<br />
| K<sub>mRNA_LuxI:antisense_mRNA</sub><br />
| 4.22E-14 [M]<br />
| Dissociation constant for complex of LuxI mRNA with antisense mRNA<br />
| [https://2008.igem.org/Team:KULeuven/Model/Memory#References [1<html>]</html>]<br />
|}<br />
<br />
===Models===<br />
==== CellDesigner([https://static.igem.org/mediawiki/2008/4/49/Memory_CellDesigner.zip SBML file])====<br />
[[Image:Memory_CellDesigner.png|600px|center|New Memory]]<br />
<br />
==== Matlab ([https://static.igem.org/mediawiki/2008/1/1f/Memory_Matlab.zip SBML file])====<br />
[[Image:Memory_Matlab.jpg|600px|center|New Memory]]<br />
<br />
=== Simulations ===<br />
<br />
<br />
Multiple simulations have been performed to see if the memory works. If we give a pulse of TetR from<br />
5E-5 to 0.0125 for 10000 sec, we see that the memory switches from its 0-state to its 1-state (left figure): <br />
cIIP22 amount drops and the cI434 increases. The high cI434 amount will repress the RNA production. If the<br />
pulse lasts for only 1000 sec, the memory remains in its 0-state (middle figure). Simulations show the<br />
minimum time span needed to switch the memory is approximately 1300 sec. The right figure shows us that a sequence<br />
of two pulses of only 1000 sec can also make the memory switch.<br />
<br />
The graphs have amounts (number of molecules in the cell) plotted vs time, measured in seconds. <br />
<br />
<html><br />
<div class="center"><br />
<div class="noborder" style="overflow: auto; width: 800px; height: 420px;"><br />
<div class="noborder" style="width: 1250px;"> <br />
<img src="https://static.igem.org/mediawiki/2008/8/80/Memory1.png" style="float: left; width: 400px; height: 400px; margin: 0 5px;" /><br />
<img src="https://static.igem.org/mediawiki/2008/3/37/Memory2.png" style="float: left; width: 400px; height: 400px; margin: 0 5px;" /><br />
<img src="https://static.igem.org/mediawiki/2008/b/bf/Memory3.png" style="float: left; width: 400px; height: 400px; margin: 0 5px;" /><br />
</div></div></div></html><br />
<br />
=== Mathematical Analysis ([https://static.igem.org/mediawiki/2008/8/87/Memory_Maple.zip Maple file])===<br />
<br />
The existence of the two stable states of the memory (cI434_LVA high and cIIP22 low / cI434_LVA low and cIIP22 high) can be mathematically proven.<br />
<br />
First we define the equilibrium points of the following differential equation system:<br />
[[Image:Differential.png|700px|center]]<br />
The equilibrium points are defined as the points for which all the derivatives are zero. Solving this non-linear system for [OmpF] equal to 0.00005 results in finding the roots of the equation <br />
[[Image:Equation.png|600px|center]]<br />
This equation has three real zeros ([CI434_LVA = 0.002435815407], [CI434_LVA = 11.52604711], [CI434_LVA = 233.6410112]) and two conjugated imaginary zeros ([CI434_LVA = -8.242012862+11.11131953*I], [CI434_LVA = -8.242012862-11.11131953*I]) as can be seen in the following figure:<br />
[[Image:Equation.jpg|300px|center]]<br />
<br />
The real roots of the system for all the variables are:<br />
{| class="wikitable" style="text-align:center"<br />
|+Real Roots<br />
|-<br />
! !! Zero 1 !! Zero 2 !! Zero 3<br />
|-<br />
! [CI434]<br />
| 1.248827120|| 1.248827120|| 1.248827120<br />
|-<br />
! [CI434-LVA]<br />
| 0.002435815407|| 11.52604711|| 233.6410112<br />
|-<br />
! [mRNA<sub>CI434-LVA</sub>]<br />
| 0.00001804584283|| 0.08539121399|| 1.730939445<br />
|-<br />
! [CIIP22]<br />
| 34.03962262|| 0.4824866257 || 0.001433763049<br />
|-<br />
! [mRNA<sub>CIIP22</sub>]<br />
| 1.765288021 || 0.02502165991 || 0.00007435466497<br />
|-<br />
! [asRNA<sub>CI434</sub>]<br />
| 0.02164042563 || 0.02164042563 || 0.02164042563<br />
|}<br />
The stability of the three real zeros is defined by the eigenvalues of the Jacobian of the differential equation system. This Jacobian equals <br />
[[Image:Jacobian.png|800px|center]]<br />
<br />
The eigenvalues for '''Zero 1''' are all negative (-0.2334056227e-2, -0.2286362516e-2, -0.3122380843e-3, -0.2645443732e-3, -0.9627000000e-4, -0.2310490600e-2). This is also the case for '''Zero 3''' (-0.2319019326e-2, -0.2301889325e-2, -0.2795812737e-3, -0.2967112750e-3, -0.9627000000e-4, -0.2310490600e-2). These two zero's represent the two stable states of the memory system. '''Zero 2''' has one positive eigenvalue (0.0002015903724) and is therefore unstable. <br />
<br />
The following figure shows some trajectories in the phase plane ([CIIP22],[CI434-LVA]): there is a clear boundary between the two stable equilibrium points (the green dots) that goes through the unstable equilibrium point (the red dot). This boundary divides the two dimensional phase plane in two separate basins of attraction.<br />
[[Image:Memory_PhasePlot.png|600px|center]]<br />
<br />
=== References ===<br />
<br />
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=utf-8"/><br />
<title>Bibliography</title><br />
</head><br />
<body><br />
<table style="border-collapse:collapse;line-height:1.1em;"><br />
<tr style="vertical-align:top;"><td>[1]</td><td style="padding-left:4pt;">A. E G H Wagner and R W Simons, “Antisense RNA Control in Bacteria, Phages, and Plasmids,” Nov. 2003; http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.mi.48.100194.003433.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[2]</td><td style="padding-left:4pt;">“ETHZ/Parameters - IGEM07”; https://2007.igem.org/ETHZ/Parameters.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<br />
<tr style="vertical-align:top;"><td>[3]</td><td style="padding-left:4pt;">J.A. Bernstein et al., “Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays,” <span style="font-style:italic;">Proceedings of the National Academy of Sciences of the United States of America</span>, vol. 99, Jul. 2002, pp. 9697–9702. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1073/pnas.112318199&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Global%20analysis%20of%20mRNA%20decay%20and%20abundance%20in%20Escherichia%20coli%20at%20single-gene%20resolution%20using%20two-color%20fluorescent%20DNA%20microarrays&amp;rft.jtitle=Proceedings%20of%20the%20National%20Academy%20of%20Sciences%20of%20the%20United%20States%20of%20America&amp;rft.stitle=Proc%20Natl%20Acad%20Sci%20U%20S%20A.%20&amp;rft.volume=99&amp;rft.issue=15&amp;rft.aufirst=Jonathan%20A.&amp;rft.aulast=Bernstein&amp;rft.au=Jonathan%20A.%20Bernstein&amp;rft.au=Arkady%20B.%20Khodursky&amp;rft.au=Pei-Hsun%20Lin&amp;rft.au=Sue%20Lin-Chao&amp;rft.au=Stanley%20N.%20Cohen&amp;rft.date=2002-07-23&amp;rft.pages=9697%E2%80%939702"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[4]</td><td style="padding-left:4pt;">M. Bon, S.J. McGowan, and P.R. Cook, “Many expressed genes in bacteria and yeast are transcribed only once per cell cycle,” <span style="font-style:italic;">FASEB J.</span>, vol. 20, Aug. 2006, pp. 1721-1723. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1096/fj.06-6087fje&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Many%20expressed%20genes%20in%20bacteria%20and%20yeast%20are%20transcribed%20only%20once%20per%20cell%20cycle&amp;rft.jtitle=FASEB%20J.&amp;rft.volume=20&amp;rft.issue=10&amp;rft.aufirst=Michael&amp;rft.aulast=Bon&amp;rft.au=Michael%20Bon&amp;rft.au=Simon%20J.%20McGowan&amp;rft.au=Peter%20R.%20Cook&amp;rft.date=2006-08-01&amp;rft.pages=1721-1723"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[5]</td><td style="padding-left:4pt;">J.B. Andersen et al., “New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria,” <span style="font-style:italic;">Applied and Environmental Microbiology</span>, vol. 64, Jun. 1998, pp. 2240–2246. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=New%20Unstable%20Variants%20of%20Green%20Fluorescent%20Protein%20for%20Studies%20of%20Transient%20Gene%20Expression%20in%20Bacteria&amp;rft.jtitle=Applied%20and%20Environmental%20Microbiology&amp;rft.stitle=Appl%20Environ%20Microbiol.%20&amp;rft.volume=64&amp;rft.issue=6&amp;rft.aufirst=Jens%20Bo&amp;rft.aulast=Andersen&amp;rft.au=Jens%20Bo%20Andersen&amp;rft.au=Claus%20Sternberg&amp;rft.au=Lars%20Kongsbak%20Poulsen&amp;rft.au=Sara%20Petersen%20Bj%C3%B8rn&amp;rft.au=Michael%20Givskov&amp;rft.au=S%C3%B8ren%20Molin&amp;rft.date=1998-06&amp;rft.pages=2240%E2%80%932246"></span></td></tr><br />
<br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[6]</td><td style="padding-left:4pt;">J. De Anda, A. Poteete, and R. Sauer, “P22 c2 repressor. Domain structure and function,” <span style="font-style:italic;">J. Biol. Chem.</span>, vol. 258, Sep. 1983, pp. 10536-10542. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=P22%20c2%20repressor.%20Domain%20structure%20and%20function&amp;rft.jtitle=J.%20Biol.%20Chem.&amp;rft.volume=258&amp;rft.issue=17&amp;rft.aufirst=J&amp;rft.aulast=De%20Anda&amp;rft.au=J%20De%20Anda&amp;rft.au=AR%20Poteete&amp;rft.au=RT%20Sauer&amp;rft.date=1983-09-10&amp;rft.pages=10536-10542"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[7]</td><td style="padding-left:4pt;">“Part:BBa B0031 - partsregistry.org”; http://partsregistry.org/wiki/index.php?title=Part:BBa_B0031.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[8]</td><td style="padding-left:4pt;">“Part:BBa B0033 - partsregistry.org”; http://partsregistry.org/Part:BBa_B0033.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[9]</td><td style="padding-left:4pt;">“Part:BBa R0040 - partsregistry.org”; http://partsregistry.org/Part:BBa_R0040.</td></tr><br />
<br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[10]</td><td style="padding-left:4pt;">“Part:BBa R0052 - partsregistry.org”; http://partsregistry.org/Part:BBa_R0052.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[11]</td><td style="padding-left:4pt;">“Part:BBa R0053 - partsregistry.org”; http://partsregistry.org/Part:BBa_R0053.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[12]</td><td style="padding-left:4pt;">B.C. McCabe, D.R. Pawlowski, and G.B. Koudelka, “The Bacteriophage 434 Repressor Dimer Preferentially Undergoes Autoproteolysis by an Intramolecular Mechanism,” <span style="font-style:italic;">J. Bacteriol.</span>, vol. 187, Aug. 2005, pp. 5624-5630. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1128/JB.187.16.5624-5630.2005&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=The%20Bacteriophage%20434%20Repressor%20Dimer%20Preferentially%20Undergoes%20Autoproteolysis%20by%20an%20Intramolecular%20Mechanism&amp;rft.jtitle=J.%20Bacteriol.&amp;rft.volume=187&amp;rft.issue=16&amp;rft.aufirst=Barbara%20C.&amp;rft.aulast=McCabe&amp;rft.au=Barbara%20C.%20McCabe&amp;rft.au=David%20R.%20Pawlowski&amp;rft.au=Gerald%20B.%20Koudelka&amp;rft.date=2005-08-15&amp;rft.pages=5624-5630"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
</table></body><br />
</html></div>BNathaliehttp://2008.igem.org/Team:KULeuven/Model/InverterTeam:KULeuven/Model/Inverter2008-10-29T23:20:08Z<p>BNathalie: /* Describing the system */</p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Scripting}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
[[Image:pictogram_inverter.png|120px|right]]<br />
<br />
==Invertimer==<br />
<br />
=== Position in the system ===<br />
<br />
The InverTimer subsystem receives its input from the filter, T7. The InverTimer's function is to '''produce''' HSL when '''no''' input is present, so a low T7 input gives rise to a high HSL output and vice versa. The production of HSL means that the cell will start a timer that eventually will be used in the celldeath-subsystem to produce ccdB. In this way the cell will die off if no desease remains present.<br />
<br />
=== Describing the system ===<br />
see also: [https://2008.igem.org/Team:KULeuven/Project/Inverter Project:InverTimer]<br />
<br />
[[Image:Inverter_BioBrick.jpg|center]]<br />
<br />
==== ODE's ====<br />
<br />
<html><br />
<body><br />
<p><br />
<a href="https://static.igem.org/mediawiki/2008/3/35/InverterODE.pdf"><br />
<img border="0" src="https://2008.igem.org/wiki/skins/common/images/icons/fileicon-pdf.png" width="65" height="60"><br />
</a><br />
</p><br />
</body><br />
</html><br />
<br />
==== Parameters ====<br />
<br />
{| width=80% style="border: 1px solid #003E81; background-color: #EEFFFF;"<br />
|+ ''Parameter values (Inverter)''<br />
! width=15% | Name<br />
! width=15% | Value<br />
! width=40% | Comments<br />
! width=10% | Reference<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Degradation Rates<br />
|- <br />
| d<sub>LuxI</sub><br />
| d<sub>LVA</sub> = 2.814E-4 s<sup>-1</sup><br />
| LVA-tag reduces lifetime to 40 minutes<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html> [7<html>]</html>]<br />
|-<br />
| d<sub>RNA_LuxI</sub><br />
| 0.0025 s<sup>-1</sup><br />
| <br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [5<html>]</html>]<br />
|-<br />
| d<sub>LuxI_antimRNA</sub><br />
| 0.0045303737 s<sup>-1</sup><br />
| estimate: because this RNA isn't translated, it degrades faster <br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [5<html>]</html>]<br />
|-<br />
| d<sub>LacI</sub><br />
| d<sub>LVA</sub> = 2.814E-4 s<sup>-1</sup><br />
| LVA-tag reduces lifetime to 40 minutes<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html> [7<html>]</html>]<br />
|-<br />
| d<sub>closed mRNA LacI</sub><br />
| 0.0046209812 s<sup>-1</sup><br />
| estimate: because this mRNA isn't translated, it degrades faster <br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [5<html>]</html>]<br />
|-<br />
| d<sub>open mRNA LacI</sub><br />
| 0.0023104906 s<sup>-1</sup><br />
| <br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [5<html>]</html>]<br />
|-<br />
| d<sub>open mRNA LacI complex</sub><br />
| 0.0023104906 s<sup>-1</sup><br />
| <br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [5<html>]</html>]<br />
|-<br />
| d<sub>HSL</sub><br />
| 1.02E-6 s<sup>-1</sup><br />
| very stable in the medium, lifetime around 185h<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [11<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | LuxI catalysis<br />
|-<br />
| k<sub>cat</sub><br />
| 0.0166666667 s<sup>-1</sup><br />
| Estimated to be about 90% of V<sub>max</sub> in LB medium.<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [4<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | T7 Transcription<br />
|-<br />
| K<sub>T7</sub><br />
| 421<br />
| dissociation constant, recalculated to remove units<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [10<html>]</html>]<br />
|-<br />
| k<sub>max</sub><br />
| 0.044 s<sup>-1</sup><br />
| maximal T7 transcription rate<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [10<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Key-Lock constants<br />
|-<br />
| K<sub>eq 1</sub><br />
| 0,015 [M]<br />
| between closed and open T7 mRNA, modeled for competition, experimental<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
| K<sub>eq 2</sub><br />
| 0.0212 [M]<br />
| between closed T7 mRNA and key unlocked mRNA complex, modeled for competition, experimental<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
| k<sub>dis2</sub><br />
| 0.00416 s<sup>-1</sup><br />
| derived from experimental values<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
| k<sub>complex2</sub><br />
| 0.00237 s<sup>-1</sup><br />
| derived from experimental values<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
| k<sub>closed</sub><br />
| 500 s<sup>-1</sup><br />
| derived from experimental values<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
| k<sub>open</sub><br />
| 7.5 s<sup>-1</sup><br />
| derived from experimental values<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | LacI repression<br />
|-<br />
| K<sub>LacI</sub><br />
| 1.0E-10 M<sup>-1</sup><br />
| Dissociation constant<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [3<html>]</html>]<br />
|- <br />
| n<sub>LacI</sub><br />
| 2.0<br />
| Hill coefficient for LacI<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [3<html>]</html>]<br />
|-<br />
| k_trans_LacI <br />
| 0.0025 s<sup>-1</sup><br />
| Estimated maximal transcription rate from R0011<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [9<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Antisense LuxI<br />
|-<br />
| k_complex3<br />
| 0.00237 s<sup>-1</sup><br />
| rate constant for formation of asRNA - LuxI mRNA duplex<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [3<html>]</html>]<br />
|-<br />
| K<sub>mRNA_LuxI:antisense_mRNA</sub><br />
| 4.22E14 <br />
| Complex of LuxI mRNA with antisense mRNA<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [1<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Translation Rates<br />
|-<br />
| k<sub>transl LuxI</sub><br />
| 0.167 s<sup>-1</sup><br />
| translation rate for B0032 RBS (0.3 relative efficiency)<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [8<html>]</html>]<br />
|-<br />
| k<sub>transl LacI</sub><br />
| 0.167 s<sup>-1</sup><br />
| estimate: lock defined translation rate for LacI<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|}<br />
<br />
=== Models ===<br />
<br />
==== CellDesigner ([https://static.igem.org/mediawiki/2008/9/9e/Inverter_CellDesigner.zip SBML file])====<br />
<br />
[[Image:Inverter_CellDesigner.png|800px|center|Inverter]]<br />
<br />
==== Matlab ([https://static.igem.org/mediawiki/2008/0/00/Inverter_Matlab.zip SBML file])====<br />
[[Image:Inverter_Matlab.jpg|700px|center|Inverter]]<br />
<br />
=== Simulations ===<br />
<br />
{| class="wikitable"<br />
|-<br />
!width="80"| Time span<br />
!width="80"| Input (TetR)<br />
!width="720"| Results<br />
|-<br />
| A<br />
| 0.0125 s<sup>-1</sup><br />
| The amount LacI increases from state zero to state one because both mRNA_RIBOKEY and pT7_tag are present. This results in a repression of LuxI which decreases to zero: the input signal (TetR) is inverted. <br />
|-<br />
| B<br />
| 5E-5 s<sup>-1</sup><br />
| The amount LacI decreases back to state zero. The amount LuxI remains the same (state zero). <br />
|-<br />
| C<br />
| 5E-5 s<sup>-1</sup><br />
| LuxI changes from state zero to state one. Time span B and C form together the transient behaviour of the inverter when the input signal changes from one to zero.<br />
|-<br />
| D<br />
| 5E-5 s<sup>-1</sup><br />
| LuxI remains in state one: the input signal is once again inverted.<br />
|-<br />
| E<br />
| 0.0125 s<sup>-1</sup><br />
| A short pulse of 1000 seconds has a influence a steep decrease of LuxI.<br />
|-<br />
| F & G<br />
| 5E-5 s<sup>-1</sup><br />
| During time span F and G, LuxI decreases further for a while and increases back to state one.<br />
|-<br />
| H<br />
| 5E-5 s<sup>-1</sup><br />
| LuxI is back in state one.<br />
|}<br />
<br />
The simulation shows a working inverter (left figure). A small disadvantage is the transient behaviour of the inverter: a small pulse of 1000 seconds results in a transient behaviour of +- 30000 seconds. Also for a long pulse (10000 seconds) is a long transient behaviour noticeable (40000 seconds). The effect of the inverter on the timer aspect is visuable in the right figure: a long pulse ( from 10000 till 11000) resets the timer (HSL drecreases till zero). After this pulse and the transient behaviour of the inverter, the timer restarts counting. The short pulse (from 200000 till 201000 seconds) only partially resets the timer.<br />
<br />
All graphs have amounts (number of molecules in the cell) plotted vs time, measured in seconds.<br />
<br />
<html><br />
<div class="center"><br />
<div class="noborder" style="overflow: auto; width: 800px; height: 520px;"><br />
<div class="noborder" style="width: 1200px;"> <br />
<img src="https://static.igem.org/mediawiki/2008/d/d1/Sim_inverter_1.png" style="float: left; width: 550px; height: 500px; margin: 0 5px;" /><br />
<img src="https://static.igem.org/mediawiki/2008/2/27/HSL.png" style="float: left; width: 600px; height: 500px; margin: 0 5px;" /><br />
</div></div></div></html><br />
<br />
=== References ===<br />
<br />
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=utf-8"/><br />
<title>Bibliography</title><br />
</head><br />
<body><br />
<table style="border-collapse:collapse;line-height:1.1em;"><br />
<tr style="vertical-align:top;"><td>[1]</td><td style="padding-left:4pt;">A. E G H Wagner and R W Simons, “Antisense RNA Control in Bacteria, Phages, and Plasmids,” Nov. 2003; http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.mi.48.100194.003433.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[2]</td><td style="padding-left:4pt;">“Berkeley2006-RiboregulatorsMain - IGEM”; http://parts2.mit.edu/wiki/index.php/Berkeley2006-RiboregulatorsMain.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<br />
<tr style="vertical-align:top;"><td>[3]</td><td style="padding-left:4pt;">“ETHZ/Parameters - IGEM07”; https://2007.igem.org/ETHZ/Parameters.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[4]</td><td style="padding-left:4pt;">“Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein,” Sep. 1996; http://www.pnas.org/content/93/18/9505.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[5]</td><td style="padding-left:4pt;">J.A. Bernstein et al., “Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays,” <span style="font-style:italic;">Proceedings of the National Academy of Sciences of the United States of America</span>, vol. 99, Jul. 2002, pp. 9697–9702. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1073/pnas.112318199&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Global%20analysis%20of%20mRNA%20decay%20and%20abundance%20in%20Escherichia%20coli%20at%20single-gene%20resolution%20using%20two-color%20fluorescent%20DNA%20microarrays&amp;rft.jtitle=Proceedings%20of%20the%20National%20Academy%20of%20Sciences%20of%20the%20United%20States%20of%20America&amp;rft.stitle=Proc%20Natl%20Acad%20Sci%20U%20S%20A.%20&amp;rft.volume=99&amp;rft.issue=15&amp;rft.aufirst=Jonathan%20A.&amp;rft.aulast=Bernstein&amp;rft.au=Jonathan%20A.%20Bernstein&amp;rft.au=Arkady%20B.%20Khodursky&amp;rft.au=Pei-Hsun%20Lin&amp;rft.au=Sue%20Lin-Chao&amp;rft.au=Stanley%20N.%20Cohen&amp;rft.date=2002-07-23&amp;rft.pages=9697%E2%80%939702"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[6]</td><td style="padding-left:4pt;">W. Hsieh et al., “Influence of sequence and distance between two operators on interaction with the lac repressor,” <span style="font-style:italic;">J. Biol. Chem.</span>, vol. 262, Oct. 1987, pp. 14583-14591. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Influence%20of%20sequence%20and%20distance%20between%20two%20operators%20on%20interaction%20with%20the%20lac%20repressor&amp;rft.jtitle=J.%20Biol.%20Chem.&amp;rft.volume=262&amp;rft.issue=30&amp;rft.aufirst=WT&amp;rft.aulast=Hsieh&amp;rft.au=WT%20Hsieh&amp;rft.au=PA%20Whitson&amp;rft.au=KS%20Matthews&amp;rft.au=RD%20Wells&amp;rft.date=1987-10-25&amp;rft.pages=14583-14591"></span></td></tr><br />
<br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[7]</td><td style="padding-left:4pt;">J.B. Andersen et al., “New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria,” <span style="font-style:italic;">Applied and Environmental Microbiology</span>, vol. 64, Jun. 1998, pp. 2240–2246. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=New%20Unstable%20Variants%20of%20Green%20Fluorescent%20Protein%20for%20Studies%20of%20Transient%20Gene%20Expression%20in%20Bacteria&amp;rft.jtitle=Applied%20and%20Environmental%20Microbiology&amp;rft.stitle=Appl%20Environ%20Microbiol.%20&amp;rft.volume=64&amp;rft.issue=6&amp;rft.aufirst=Jens%20Bo&amp;rft.aulast=Andersen&amp;rft.au=Jens%20Bo%20Andersen&amp;rft.au=Claus%20Sternberg&amp;rft.au=Lars%20Kongsbak%20Poulsen&amp;rft.au=Sara%20Petersen%20Bj%C3%B8rn&amp;rft.au=Michael%20Givskov&amp;rft.au=S%C3%B8ren%20Molin&amp;rft.date=1998-06&amp;rft.pages=2240%E2%80%932246"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[8]</td><td style="padding-left:4pt;">“Part:BBa B0032 - partsregistry.org”; http://partsregistry.org/Part:BBa_B0032.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[9]</td><td style="padding-left:4pt;">“Part:BBa R0011 - partsregistry.org”; http://partsregistry.org/Part:BBa_R0011.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[10]</td><td style="padding-left:4pt;">G.M. Skinner et al., “Promoter Binding, Initiation, and Elongation By Bacteriophage T7 RNA Polymerase: A SINGLE-MOLECULE VIEW OF THE TRANSCRIPTION CYCLE,” <span style="font-style:italic;">J. Biol. Chem.</span>, vol. 279, Jan. 2004, pp. 3239-3244. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1074/jbc.M310471200&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Promoter%20Binding%2C%20Initiation%2C%20and%20Elongation%20By%20Bacteriophage%20T7%20RNA%20Polymerase%3A%20A%20SINGLE-MOLECULE%20VIEW%20OF%20THE%20TRANSCRIPTION%20CYCLE&amp;rft.jtitle=J.%20Biol.%20Chem.&amp;rft.volume=279&amp;rft.issue=5&amp;rft.aufirst=Gary%20M.&amp;rft.aulast=Skinner&amp;rft.au=Gary%20M.%20Skinner&amp;rft.au=Christoph%20G.%20Baumann&amp;rft.au=Diana%20M.%20Quinn&amp;rft.au=Justin%20E.%20Molloy&amp;rft.au=James%20G.%20Hoggett&amp;rft.date=2004&amp;rft.pages=3239-3244"></span></td></tr><br />
<br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[11]</td><td style="padding-left:4pt;">Y. Wang and J.R. Leadbetter, “Rapid Acyl-Homoserine Lactone Quorum Signal Biodegradation in Diverse Soils,” <span style="font-style:italic;">Appl. Environ. Microbiol.</span>, vol. 71, Mar. 2005, pp. 1291-1299. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1128/AEM.71.3.1291-1299.2005&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Rapid%20Acyl-Homoserine%20Lactone%20Quorum%20Signal%20Biodegradation%20in%20Diverse%20Soils&amp;rft.jtitle=Appl.%20Environ.%20Microbiol.&amp;rft.volume=71&amp;rft.issue=3&amp;rft.aufirst=Ya-Juan&amp;rft.aulast=Wang&amp;rft.au=Ya-Juan%20Wang&amp;rft.au=Jared%20Renton%20Leadbetter&amp;rft.date=2005-03-01&amp;rft.pages=1291-1299"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
</table></body><br />
</html></div>BNathaliehttp://2008.igem.org/Team:KULeuven/Model/InverterTeam:KULeuven/Model/Inverter2008-10-29T23:19:48Z<p>BNathalie: /* Position in the system */</p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Scripting}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
[[Image:pictogram_inverter.png|120px|right]]<br />
<br />
==Invertimer==<br />
<br />
=== Position in the system ===<br />
<br />
The InverTimer subsystem receives its input from the filter, T7. The InverTimer's function is to '''produce''' HSL when '''no''' input is present, so a low T7 input gives rise to a high HSL output and vice versa. The production of HSL means that the cell will start a timer that eventually will be used in the celldeath-subsystem to produce ccdB. In this way the cell will die off if no desease remains present.<br />
<br />
=== Describing the system ===<br />
see also: [https://2008.igem.org/Team:KULeuven/Project/Inverter Project:Invertimer]<br />
<br />
[[Image:Inverter_BioBrick.jpg|center]]<br />
<br />
==== ODE's ====<br />
<br />
<html><br />
<body><br />
<p><br />
<a href="https://static.igem.org/mediawiki/2008/3/35/InverterODE.pdf"><br />
<img border="0" src="https://2008.igem.org/wiki/skins/common/images/icons/fileicon-pdf.png" width="65" height="60"><br />
</a><br />
</p><br />
</body><br />
</html><br />
<br />
==== Parameters ====<br />
<br />
{| width=80% style="border: 1px solid #003E81; background-color: #EEFFFF;"<br />
|+ ''Parameter values (Inverter)''<br />
! width=15% | Name<br />
! width=15% | Value<br />
! width=40% | Comments<br />
! width=10% | Reference<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Degradation Rates<br />
|- <br />
| d<sub>LuxI</sub><br />
| d<sub>LVA</sub> = 2.814E-4 s<sup>-1</sup><br />
| LVA-tag reduces lifetime to 40 minutes<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html> [7<html>]</html>]<br />
|-<br />
| d<sub>RNA_LuxI</sub><br />
| 0.0025 s<sup>-1</sup><br />
| <br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [5<html>]</html>]<br />
|-<br />
| d<sub>LuxI_antimRNA</sub><br />
| 0.0045303737 s<sup>-1</sup><br />
| estimate: because this RNA isn't translated, it degrades faster <br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [5<html>]</html>]<br />
|-<br />
| d<sub>LacI</sub><br />
| d<sub>LVA</sub> = 2.814E-4 s<sup>-1</sup><br />
| LVA-tag reduces lifetime to 40 minutes<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html> [7<html>]</html>]<br />
|-<br />
| d<sub>closed mRNA LacI</sub><br />
| 0.0046209812 s<sup>-1</sup><br />
| estimate: because this mRNA isn't translated, it degrades faster <br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [5<html>]</html>]<br />
|-<br />
| d<sub>open mRNA LacI</sub><br />
| 0.0023104906 s<sup>-1</sup><br />
| <br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [5<html>]</html>]<br />
|-<br />
| d<sub>open mRNA LacI complex</sub><br />
| 0.0023104906 s<sup>-1</sup><br />
| <br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [5<html>]</html>]<br />
|-<br />
| d<sub>HSL</sub><br />
| 1.02E-6 s<sup>-1</sup><br />
| very stable in the medium, lifetime around 185h<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [11<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | LuxI catalysis<br />
|-<br />
| k<sub>cat</sub><br />
| 0.0166666667 s<sup>-1</sup><br />
| Estimated to be about 90% of V<sub>max</sub> in LB medium.<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [4<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | T7 Transcription<br />
|-<br />
| K<sub>T7</sub><br />
| 421<br />
| dissociation constant, recalculated to remove units<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [10<html>]</html>]<br />
|-<br />
| k<sub>max</sub><br />
| 0.044 s<sup>-1</sup><br />
| maximal T7 transcription rate<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [10<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Key-Lock constants<br />
|-<br />
| K<sub>eq 1</sub><br />
| 0,015 [M]<br />
| between closed and open T7 mRNA, modeled for competition, experimental<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
| K<sub>eq 2</sub><br />
| 0.0212 [M]<br />
| between closed T7 mRNA and key unlocked mRNA complex, modeled for competition, experimental<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
| k<sub>dis2</sub><br />
| 0.00416 s<sup>-1</sup><br />
| derived from experimental values<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
| k<sub>complex2</sub><br />
| 0.00237 s<sup>-1</sup><br />
| derived from experimental values<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
| k<sub>closed</sub><br />
| 500 s<sup>-1</sup><br />
| derived from experimental values<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
| k<sub>open</sub><br />
| 7.5 s<sup>-1</sup><br />
| derived from experimental values<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | LacI repression<br />
|-<br />
| K<sub>LacI</sub><br />
| 1.0E-10 M<sup>-1</sup><br />
| Dissociation constant<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [3<html>]</html>]<br />
|- <br />
| n<sub>LacI</sub><br />
| 2.0<br />
| Hill coefficient for LacI<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [3<html>]</html>]<br />
|-<br />
| k_trans_LacI <br />
| 0.0025 s<sup>-1</sup><br />
| Estimated maximal transcription rate from R0011<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [9<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Antisense LuxI<br />
|-<br />
| k_complex3<br />
| 0.00237 s<sup>-1</sup><br />
| rate constant for formation of asRNA - LuxI mRNA duplex<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [3<html>]</html>]<br />
|-<br />
| K<sub>mRNA_LuxI:antisense_mRNA</sub><br />
| 4.22E14 <br />
| Complex of LuxI mRNA with antisense mRNA<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [1<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Translation Rates<br />
|-<br />
| k<sub>transl LuxI</sub><br />
| 0.167 s<sup>-1</sup><br />
| translation rate for B0032 RBS (0.3 relative efficiency)<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [8<html>]</html>]<br />
|-<br />
| k<sub>transl LacI</sub><br />
| 0.167 s<sup>-1</sup><br />
| estimate: lock defined translation rate for LacI<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|}<br />
<br />
=== Models ===<br />
<br />
==== CellDesigner ([https://static.igem.org/mediawiki/2008/9/9e/Inverter_CellDesigner.zip SBML file])====<br />
<br />
[[Image:Inverter_CellDesigner.png|800px|center|Inverter]]<br />
<br />
==== Matlab ([https://static.igem.org/mediawiki/2008/0/00/Inverter_Matlab.zip SBML file])====<br />
[[Image:Inverter_Matlab.jpg|700px|center|Inverter]]<br />
<br />
=== Simulations ===<br />
<br />
{| class="wikitable"<br />
|-<br />
!width="80"| Time span<br />
!width="80"| Input (TetR)<br />
!width="720"| Results<br />
|-<br />
| A<br />
| 0.0125 s<sup>-1</sup><br />
| The amount LacI increases from state zero to state one because both mRNA_RIBOKEY and pT7_tag are present. This results in a repression of LuxI which decreases to zero: the input signal (TetR) is inverted. <br />
|-<br />
| B<br />
| 5E-5 s<sup>-1</sup><br />
| The amount LacI decreases back to state zero. The amount LuxI remains the same (state zero). <br />
|-<br />
| C<br />
| 5E-5 s<sup>-1</sup><br />
| LuxI changes from state zero to state one. Time span B and C form together the transient behaviour of the inverter when the input signal changes from one to zero.<br />
|-<br />
| D<br />
| 5E-5 s<sup>-1</sup><br />
| LuxI remains in state one: the input signal is once again inverted.<br />
|-<br />
| E<br />
| 0.0125 s<sup>-1</sup><br />
| A short pulse of 1000 seconds has a influence a steep decrease of LuxI.<br />
|-<br />
| F & G<br />
| 5E-5 s<sup>-1</sup><br />
| During time span F and G, LuxI decreases further for a while and increases back to state one.<br />
|-<br />
| H<br />
| 5E-5 s<sup>-1</sup><br />
| LuxI is back in state one.<br />
|}<br />
<br />
The simulation shows a working inverter (left figure). A small disadvantage is the transient behaviour of the inverter: a small pulse of 1000 seconds results in a transient behaviour of +- 30000 seconds. Also for a long pulse (10000 seconds) is a long transient behaviour noticeable (40000 seconds). The effect of the inverter on the timer aspect is visuable in the right figure: a long pulse ( from 10000 till 11000) resets the timer (HSL drecreases till zero). After this pulse and the transient behaviour of the inverter, the timer restarts counting. The short pulse (from 200000 till 201000 seconds) only partially resets the timer.<br />
<br />
All graphs have amounts (number of molecules in the cell) plotted vs time, measured in seconds.<br />
<br />
<html><br />
<div class="center"><br />
<div class="noborder" style="overflow: auto; width: 800px; height: 520px;"><br />
<div class="noborder" style="width: 1200px;"> <br />
<img src="https://static.igem.org/mediawiki/2008/d/d1/Sim_inverter_1.png" style="float: left; width: 550px; height: 500px; margin: 0 5px;" /><br />
<img src="https://static.igem.org/mediawiki/2008/2/27/HSL.png" style="float: left; width: 600px; height: 500px; margin: 0 5px;" /><br />
</div></div></div></html><br />
<br />
=== References ===<br />
<br />
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=utf-8"/><br />
<title>Bibliography</title><br />
</head><br />
<body><br />
<table style="border-collapse:collapse;line-height:1.1em;"><br />
<tr style="vertical-align:top;"><td>[1]</td><td style="padding-left:4pt;">A. E G H Wagner and R W Simons, “Antisense RNA Control in Bacteria, Phages, and Plasmids,” Nov. 2003; http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.mi.48.100194.003433.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[2]</td><td style="padding-left:4pt;">“Berkeley2006-RiboregulatorsMain - IGEM”; http://parts2.mit.edu/wiki/index.php/Berkeley2006-RiboregulatorsMain.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<br />
<tr style="vertical-align:top;"><td>[3]</td><td style="padding-left:4pt;">“ETHZ/Parameters - IGEM07”; http://parts.mit.edu/igem07/index.php/ETHZ/Parameters.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[4]</td><td style="padding-left:4pt;">“Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein,” Sep. 1996; http://www.pnas.org/content/93/18/9505.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[5]</td><td style="padding-left:4pt;">J.A. Bernstein et al., “Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays,” <span style="font-style:italic;">Proceedings of the National Academy of Sciences of the United States of America</span>, vol. 99, Jul. 2002, pp. 9697–9702. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1073/pnas.112318199&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Global%20analysis%20of%20mRNA%20decay%20and%20abundance%20in%20Escherichia%20coli%20at%20single-gene%20resolution%20using%20two-color%20fluorescent%20DNA%20microarrays&amp;rft.jtitle=Proceedings%20of%20the%20National%20Academy%20of%20Sciences%20of%20the%20United%20States%20of%20America&amp;rft.stitle=Proc%20Natl%20Acad%20Sci%20U%20S%20A.%20&amp;rft.volume=99&amp;rft.issue=15&amp;rft.aufirst=Jonathan%20A.&amp;rft.aulast=Bernstein&amp;rft.au=Jonathan%20A.%20Bernstein&amp;rft.au=Arkady%20B.%20Khodursky&amp;rft.au=Pei-Hsun%20Lin&amp;rft.au=Sue%20Lin-Chao&amp;rft.au=Stanley%20N.%20Cohen&amp;rft.date=2002-07-23&amp;rft.pages=9697%E2%80%939702"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[6]</td><td style="padding-left:4pt;">W. Hsieh et al., “Influence of sequence and distance between two operators on interaction with the lac repressor,” <span style="font-style:italic;">J. Biol. Chem.</span>, vol. 262, Oct. 1987, pp. 14583-14591. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Influence%20of%20sequence%20and%20distance%20between%20two%20operators%20on%20interaction%20with%20the%20lac%20repressor&amp;rft.jtitle=J.%20Biol.%20Chem.&amp;rft.volume=262&amp;rft.issue=30&amp;rft.aufirst=WT&amp;rft.aulast=Hsieh&amp;rft.au=WT%20Hsieh&amp;rft.au=PA%20Whitson&amp;rft.au=KS%20Matthews&amp;rft.au=RD%20Wells&amp;rft.date=1987-10-25&amp;rft.pages=14583-14591"></span></td></tr><br />
<br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[7]</td><td style="padding-left:4pt;">J.B. Andersen et al., “New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria,” <span style="font-style:italic;">Applied and Environmental Microbiology</span>, vol. 64, Jun. 1998, pp. 2240–2246. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=New%20Unstable%20Variants%20of%20Green%20Fluorescent%20Protein%20for%20Studies%20of%20Transient%20Gene%20Expression%20in%20Bacteria&amp;rft.jtitle=Applied%20and%20Environmental%20Microbiology&amp;rft.stitle=Appl%20Environ%20Microbiol.%20&amp;rft.volume=64&amp;rft.issue=6&amp;rft.aufirst=Jens%20Bo&amp;rft.aulast=Andersen&amp;rft.au=Jens%20Bo%20Andersen&amp;rft.au=Claus%20Sternberg&amp;rft.au=Lars%20Kongsbak%20Poulsen&amp;rft.au=Sara%20Petersen%20Bj%C3%B8rn&amp;rft.au=Michael%20Givskov&amp;rft.au=S%C3%B8ren%20Molin&amp;rft.date=1998-06&amp;rft.pages=2240%E2%80%932246"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[8]</td><td style="padding-left:4pt;">“Part:BBa B0032 - partsregistry.org”; http://partsregistry.org/Part:BBa_B0032.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[9]</td><td style="padding-left:4pt;">“Part:BBa R0011 - partsregistry.org”; http://partsregistry.org/Part:BBa_R0011.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[10]</td><td style="padding-left:4pt;">G.M. Skinner et al., “Promoter Binding, Initiation, and Elongation By Bacteriophage T7 RNA Polymerase: A SINGLE-MOLECULE VIEW OF THE TRANSCRIPTION CYCLE,” <span style="font-style:italic;">J. Biol. Chem.</span>, vol. 279, Jan. 2004, pp. 3239-3244. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1074/jbc.M310471200&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Promoter%20Binding%2C%20Initiation%2C%20and%20Elongation%20By%20Bacteriophage%20T7%20RNA%20Polymerase%3A%20A%20SINGLE-MOLECULE%20VIEW%20OF%20THE%20TRANSCRIPTION%20CYCLE&amp;rft.jtitle=J.%20Biol.%20Chem.&amp;rft.volume=279&amp;rft.issue=5&amp;rft.aufirst=Gary%20M.&amp;rft.aulast=Skinner&amp;rft.au=Gary%20M.%20Skinner&amp;rft.au=Christoph%20G.%20Baumann&amp;rft.au=Diana%20M.%20Quinn&amp;rft.au=Justin%20E.%20Molloy&amp;rft.au=James%20G.%20Hoggett&amp;rft.date=2004&amp;rft.pages=3239-3244"></span></td></tr><br />
<br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[11]</td><td style="padding-left:4pt;">Y. Wang and J.R. Leadbetter, “Rapid Acyl-Homoserine Lactone Quorum Signal Biodegradation in Diverse Soils,” <span style="font-style:italic;">Appl. Environ. Microbiol.</span>, vol. 71, Mar. 2005, pp. 1291-1299. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1128/AEM.71.3.1291-1299.2005&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Rapid%20Acyl-Homoserine%20Lactone%20Quorum%20Signal%20Biodegradation%20in%20Diverse%20Soils&amp;rft.jtitle=Appl.%20Environ.%20Microbiol.&amp;rft.volume=71&amp;rft.issue=3&amp;rft.aufirst=Ya-Juan&amp;rft.aulast=Wang&amp;rft.au=Ya-Juan%20Wang&amp;rft.au=Jared%20Renton%20Leadbetter&amp;rft.date=2005-03-01&amp;rft.pages=1291-1299"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
</table></body><br />
</html></div>BNathaliehttp://2008.igem.org/Team:KULeuven/Model/InverterTeam:KULeuven/Model/Inverter2008-10-29T23:19:04Z<p>BNathalie: </p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Scripting}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
[[Image:pictogram_inverter.png|120px|right]]<br />
<br />
==Invertimer==<br />
<br />
=== Position in the system ===<br />
<br />
The invertimer subsystem receives its input from the filter, T7. The invertimer's function is to '''produce''' HSL when '''no''' input is present, so a low T7 input gives rise to a high HSL output and vice versa. The production of HSL means that the cell will start a timer that eventually will be used in the celldeath-subsystem to produce ccdB. In this way the cell will die off if no desease remains present.<br />
<br />
=== Describing the system ===<br />
see also: [https://2008.igem.org/Team:KULeuven/Project/Inverter Project:Invertimer]<br />
<br />
[[Image:Inverter_BioBrick.jpg|center]]<br />
<br />
==== ODE's ====<br />
<br />
<html><br />
<body><br />
<p><br />
<a href="https://static.igem.org/mediawiki/2008/3/35/InverterODE.pdf"><br />
<img border="0" src="https://2008.igem.org/wiki/skins/common/images/icons/fileicon-pdf.png" width="65" height="60"><br />
</a><br />
</p><br />
</body><br />
</html><br />
<br />
==== Parameters ====<br />
<br />
{| width=80% style="border: 1px solid #003E81; background-color: #EEFFFF;"<br />
|+ ''Parameter values (Inverter)''<br />
! width=15% | Name<br />
! width=15% | Value<br />
! width=40% | Comments<br />
! width=10% | Reference<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Degradation Rates<br />
|- <br />
| d<sub>LuxI</sub><br />
| d<sub>LVA</sub> = 2.814E-4 s<sup>-1</sup><br />
| LVA-tag reduces lifetime to 40 minutes<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html> [7<html>]</html>]<br />
|-<br />
| d<sub>RNA_LuxI</sub><br />
| 0.0025 s<sup>-1</sup><br />
| <br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [5<html>]</html>]<br />
|-<br />
| d<sub>LuxI_antimRNA</sub><br />
| 0.0045303737 s<sup>-1</sup><br />
| estimate: because this RNA isn't translated, it degrades faster <br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [5<html>]</html>]<br />
|-<br />
| d<sub>LacI</sub><br />
| d<sub>LVA</sub> = 2.814E-4 s<sup>-1</sup><br />
| LVA-tag reduces lifetime to 40 minutes<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html> [7<html>]</html>]<br />
|-<br />
| d<sub>closed mRNA LacI</sub><br />
| 0.0046209812 s<sup>-1</sup><br />
| estimate: because this mRNA isn't translated, it degrades faster <br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [5<html>]</html>]<br />
|-<br />
| d<sub>open mRNA LacI</sub><br />
| 0.0023104906 s<sup>-1</sup><br />
| <br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [5<html>]</html>]<br />
|-<br />
| d<sub>open mRNA LacI complex</sub><br />
| 0.0023104906 s<sup>-1</sup><br />
| <br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [5<html>]</html>]<br />
|-<br />
| d<sub>HSL</sub><br />
| 1.02E-6 s<sup>-1</sup><br />
| very stable in the medium, lifetime around 185h<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [11<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | LuxI catalysis<br />
|-<br />
| k<sub>cat</sub><br />
| 0.0166666667 s<sup>-1</sup><br />
| Estimated to be about 90% of V<sub>max</sub> in LB medium.<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [4<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | T7 Transcription<br />
|-<br />
| K<sub>T7</sub><br />
| 421<br />
| dissociation constant, recalculated to remove units<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [10<html>]</html>]<br />
|-<br />
| k<sub>max</sub><br />
| 0.044 s<sup>-1</sup><br />
| maximal T7 transcription rate<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [10<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Key-Lock constants<br />
|-<br />
| K<sub>eq 1</sub><br />
| 0,015 [M]<br />
| between closed and open T7 mRNA, modeled for competition, experimental<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
| K<sub>eq 2</sub><br />
| 0.0212 [M]<br />
| between closed T7 mRNA and key unlocked mRNA complex, modeled for competition, experimental<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
| k<sub>dis2</sub><br />
| 0.00416 s<sup>-1</sup><br />
| derived from experimental values<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
| k<sub>complex2</sub><br />
| 0.00237 s<sup>-1</sup><br />
| derived from experimental values<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
| k<sub>closed</sub><br />
| 500 s<sup>-1</sup><br />
| derived from experimental values<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
| k<sub>open</sub><br />
| 7.5 s<sup>-1</sup><br />
| derived from experimental values<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | LacI repression<br />
|-<br />
| K<sub>LacI</sub><br />
| 1.0E-10 M<sup>-1</sup><br />
| Dissociation constant<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [3<html>]</html>]<br />
|- <br />
| n<sub>LacI</sub><br />
| 2.0<br />
| Hill coefficient for LacI<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [3<html>]</html>]<br />
|-<br />
| k_trans_LacI <br />
| 0.0025 s<sup>-1</sup><br />
| Estimated maximal transcription rate from R0011<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [9<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Antisense LuxI<br />
|-<br />
| k_complex3<br />
| 0.00237 s<sup>-1</sup><br />
| rate constant for formation of asRNA - LuxI mRNA duplex<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [3<html>]</html>]<br />
|-<br />
| K<sub>mRNA_LuxI:antisense_mRNA</sub><br />
| 4.22E14 <br />
| Complex of LuxI mRNA with antisense mRNA<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [1<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Translation Rates<br />
|-<br />
| k<sub>transl LuxI</sub><br />
| 0.167 s<sup>-1</sup><br />
| translation rate for B0032 RBS (0.3 relative efficiency)<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [8<html>]</html>]<br />
|-<br />
| k<sub>transl LacI</sub><br />
| 0.167 s<sup>-1</sup><br />
| estimate: lock defined translation rate for LacI<br />
| [https://2008.igem.org/Team:KULeuven/Model/Inverter#References [2<html>]</html>]<br />
|}<br />
<br />
=== Models ===<br />
<br />
==== CellDesigner ([https://static.igem.org/mediawiki/2008/9/9e/Inverter_CellDesigner.zip SBML file])====<br />
<br />
[[Image:Inverter_CellDesigner.png|800px|center|Inverter]]<br />
<br />
==== Matlab ([https://static.igem.org/mediawiki/2008/0/00/Inverter_Matlab.zip SBML file])====<br />
[[Image:Inverter_Matlab.jpg|700px|center|Inverter]]<br />
<br />
=== Simulations ===<br />
<br />
{| class="wikitable"<br />
|-<br />
!width="80"| Time span<br />
!width="80"| Input (TetR)<br />
!width="720"| Results<br />
|-<br />
| A<br />
| 0.0125 s<sup>-1</sup><br />
| The amount LacI increases from state zero to state one because both mRNA_RIBOKEY and pT7_tag are present. This results in a repression of LuxI which decreases to zero: the input signal (TetR) is inverted. <br />
|-<br />
| B<br />
| 5E-5 s<sup>-1</sup><br />
| The amount LacI decreases back to state zero. The amount LuxI remains the same (state zero). <br />
|-<br />
| C<br />
| 5E-5 s<sup>-1</sup><br />
| LuxI changes from state zero to state one. Time span B and C form together the transient behaviour of the inverter when the input signal changes from one to zero.<br />
|-<br />
| D<br />
| 5E-5 s<sup>-1</sup><br />
| LuxI remains in state one: the input signal is once again inverted.<br />
|-<br />
| E<br />
| 0.0125 s<sup>-1</sup><br />
| A short pulse of 1000 seconds has a influence a steep decrease of LuxI.<br />
|-<br />
| F & G<br />
| 5E-5 s<sup>-1</sup><br />
| During time span F and G, LuxI decreases further for a while and increases back to state one.<br />
|-<br />
| H<br />
| 5E-5 s<sup>-1</sup><br />
| LuxI is back in state one.<br />
|}<br />
<br />
The simulation shows a working inverter (left figure). A small disadvantage is the transient behaviour of the inverter: a small pulse of 1000 seconds results in a transient behaviour of +- 30000 seconds. Also for a long pulse (10000 seconds) is a long transient behaviour noticeable (40000 seconds). The effect of the inverter on the timer aspect is visuable in the right figure: a long pulse ( from 10000 till 11000) resets the timer (HSL drecreases till zero). After this pulse and the transient behaviour of the inverter, the timer restarts counting. The short pulse (from 200000 till 201000 seconds) only partially resets the timer.<br />
<br />
All graphs have amounts (number of molecules in the cell) plotted vs time, measured in seconds.<br />
<br />
<html><br />
<div class="center"><br />
<div class="noborder" style="overflow: auto; width: 800px; height: 520px;"><br />
<div class="noborder" style="width: 1200px;"> <br />
<img src="https://static.igem.org/mediawiki/2008/d/d1/Sim_inverter_1.png" style="float: left; width: 550px; height: 500px; margin: 0 5px;" /><br />
<img src="https://static.igem.org/mediawiki/2008/2/27/HSL.png" style="float: left; width: 600px; height: 500px; margin: 0 5px;" /><br />
</div></div></div></html><br />
<br />
=== References ===<br />
<br />
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=utf-8"/><br />
<title>Bibliography</title><br />
</head><br />
<body><br />
<table style="border-collapse:collapse;line-height:1.1em;"><br />
<tr style="vertical-align:top;"><td>[1]</td><td style="padding-left:4pt;">A. E G H Wagner and R W Simons, “Antisense RNA Control in Bacteria, Phages, and Plasmids,” Nov. 2003; http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.mi.48.100194.003433.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[2]</td><td style="padding-left:4pt;">“Berkeley2006-RiboregulatorsMain - IGEM”; http://parts2.mit.edu/wiki/index.php/Berkeley2006-RiboregulatorsMain.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<br />
<tr style="vertical-align:top;"><td>[3]</td><td style="padding-left:4pt;">“ETHZ/Parameters - IGEM07”; http://parts.mit.edu/igem07/index.php/ETHZ/Parameters.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[4]</td><td style="padding-left:4pt;">“Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein,” Sep. 1996; http://www.pnas.org/content/93/18/9505.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[5]</td><td style="padding-left:4pt;">J.A. Bernstein et al., “Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays,” <span style="font-style:italic;">Proceedings of the National Academy of Sciences of the United States of America</span>, vol. 99, Jul. 2002, pp. 9697–9702. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1073/pnas.112318199&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Global%20analysis%20of%20mRNA%20decay%20and%20abundance%20in%20Escherichia%20coli%20at%20single-gene%20resolution%20using%20two-color%20fluorescent%20DNA%20microarrays&amp;rft.jtitle=Proceedings%20of%20the%20National%20Academy%20of%20Sciences%20of%20the%20United%20States%20of%20America&amp;rft.stitle=Proc%20Natl%20Acad%20Sci%20U%20S%20A.%20&amp;rft.volume=99&amp;rft.issue=15&amp;rft.aufirst=Jonathan%20A.&amp;rft.aulast=Bernstein&amp;rft.au=Jonathan%20A.%20Bernstein&amp;rft.au=Arkady%20B.%20Khodursky&amp;rft.au=Pei-Hsun%20Lin&amp;rft.au=Sue%20Lin-Chao&amp;rft.au=Stanley%20N.%20Cohen&amp;rft.date=2002-07-23&amp;rft.pages=9697%E2%80%939702"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[6]</td><td style="padding-left:4pt;">W. Hsieh et al., “Influence of sequence and distance between two operators on interaction with the lac repressor,” <span style="font-style:italic;">J. Biol. Chem.</span>, vol. 262, Oct. 1987, pp. 14583-14591. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Influence%20of%20sequence%20and%20distance%20between%20two%20operators%20on%20interaction%20with%20the%20lac%20repressor&amp;rft.jtitle=J.%20Biol.%20Chem.&amp;rft.volume=262&amp;rft.issue=30&amp;rft.aufirst=WT&amp;rft.aulast=Hsieh&amp;rft.au=WT%20Hsieh&amp;rft.au=PA%20Whitson&amp;rft.au=KS%20Matthews&amp;rft.au=RD%20Wells&amp;rft.date=1987-10-25&amp;rft.pages=14583-14591"></span></td></tr><br />
<br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[7]</td><td style="padding-left:4pt;">J.B. Andersen et al., “New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria,” <span style="font-style:italic;">Applied and Environmental Microbiology</span>, vol. 64, Jun. 1998, pp. 2240–2246. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=New%20Unstable%20Variants%20of%20Green%20Fluorescent%20Protein%20for%20Studies%20of%20Transient%20Gene%20Expression%20in%20Bacteria&amp;rft.jtitle=Applied%20and%20Environmental%20Microbiology&amp;rft.stitle=Appl%20Environ%20Microbiol.%20&amp;rft.volume=64&amp;rft.issue=6&amp;rft.aufirst=Jens%20Bo&amp;rft.aulast=Andersen&amp;rft.au=Jens%20Bo%20Andersen&amp;rft.au=Claus%20Sternberg&amp;rft.au=Lars%20Kongsbak%20Poulsen&amp;rft.au=Sara%20Petersen%20Bj%C3%B8rn&amp;rft.au=Michael%20Givskov&amp;rft.au=S%C3%B8ren%20Molin&amp;rft.date=1998-06&amp;rft.pages=2240%E2%80%932246"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[8]</td><td style="padding-left:4pt;">“Part:BBa B0032 - partsregistry.org”; http://partsregistry.org/Part:BBa_B0032.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[9]</td><td style="padding-left:4pt;">“Part:BBa R0011 - partsregistry.org”; http://partsregistry.org/Part:BBa_R0011.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[10]</td><td style="padding-left:4pt;">G.M. Skinner et al., “Promoter Binding, Initiation, and Elongation By Bacteriophage T7 RNA Polymerase: A SINGLE-MOLECULE VIEW OF THE TRANSCRIPTION CYCLE,” <span style="font-style:italic;">J. Biol. Chem.</span>, vol. 279, Jan. 2004, pp. 3239-3244. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1074/jbc.M310471200&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Promoter%20Binding%2C%20Initiation%2C%20and%20Elongation%20By%20Bacteriophage%20T7%20RNA%20Polymerase%3A%20A%20SINGLE-MOLECULE%20VIEW%20OF%20THE%20TRANSCRIPTION%20CYCLE&amp;rft.jtitle=J.%20Biol.%20Chem.&amp;rft.volume=279&amp;rft.issue=5&amp;rft.aufirst=Gary%20M.&amp;rft.aulast=Skinner&amp;rft.au=Gary%20M.%20Skinner&amp;rft.au=Christoph%20G.%20Baumann&amp;rft.au=Diana%20M.%20Quinn&amp;rft.au=Justin%20E.%20Molloy&amp;rft.au=James%20G.%20Hoggett&amp;rft.date=2004&amp;rft.pages=3239-3244"></span></td></tr><br />
<br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[11]</td><td style="padding-left:4pt;">Y. Wang and J.R. Leadbetter, “Rapid Acyl-Homoserine Lactone Quorum Signal Biodegradation in Diverse Soils,” <span style="font-style:italic;">Appl. Environ. Microbiol.</span>, vol. 71, Mar. 2005, pp. 1291-1299. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1128/AEM.71.3.1291-1299.2005&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Rapid%20Acyl-Homoserine%20Lactone%20Quorum%20Signal%20Biodegradation%20in%20Diverse%20Soils&amp;rft.jtitle=Appl.%20Environ.%20Microbiol.&amp;rft.volume=71&amp;rft.issue=3&amp;rft.aufirst=Ya-Juan&amp;rft.aulast=Wang&amp;rft.au=Ya-Juan%20Wang&amp;rft.au=Jared%20Renton%20Leadbetter&amp;rft.date=2005-03-01&amp;rft.pages=1291-1299"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
</table></body><br />
</html></div>BNathaliehttp://2008.igem.org/Team:KULeuven/Model/FilterTeam:KULeuven/Model/Filter2008-10-29T23:12:58Z<p>BNathalie: </p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Scripting}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
[[Image:pictogram_filter.png|120px|right]]<br />
<br />
== Filter ==<br />
<br />
=== Position in the system ===<br />
<br />
The filter is positioned immediately after the input, because its job is to filter out possible noise signals or background signals that aren't caused by the "desease". It is the starting piece of the whole system, situated before the invertimer- and the reset-subsystem.<br />
<br />
=== Describing the system ===<br />
see also: [https://2008.igem.org/Team:KULeuven/Project/Filter Project:Filter]<br />
<br />
[[Image:Filter_BioBrick.jpg|center]]<br />
<br />
==== ODE's ====<br />
<br />
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<p><br />
<a href="https://static.igem.org/mediawiki/2008/2/2a/FilterODE.pdff"><br />
<img border="0" src="https://2008.igem.org/wiki/skins/common/images/icons/fileicon-pdf.png" width="65" height="60"><br />
</a><br />
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<br />
==== Parameters ====<br />
<br />
{| width=80% style="border: 1px solid #003E81; background-color: #EEFFFF;"<br />
|+ ''Parameter values Filter''<br />
! width=15% | Name<br />
! width=15% | Value<br />
! width=40% | Comments<br />
! width=10% | Reference<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Degradation rates<br />
|-<br />
| d<sub>pT7 tag</sub><br />
| 0.00155 s<sup>-1</sup><br />
| UmuD tag added to speed up degradation of otherwise too stable T7 polymerase<br />
| [https://2008.igem.org/Team:KULeuven/Model/Filter#References [4<html>]</html> [5<html>]</html> [8<html>]</html>]<br />
|-<br />
| d<sub>mRNA RIBOKEY</sub><br />
| 0.00462 s<sup>-1</sup><br />
| estimate: because this RNA isn't translated, it degrades faster<br />
|[https://2008.igem.org/Team:KULeuven/Model/Filter#References [3<html>]</html>]<br />
|-<br />
| d<sub>closed mRNA T7</sub><br />
| 0.00462 s<sup>-1</sup><br />
| estimate: because this mRNA isn't translated, it degrades faster<br />
|[https://2008.igem.org/Team:KULeuven/Model/Filter#References [3<html>]</html>]<br />
|-<br />
| d<sub>open mRNA T7</sub><br />
| 0.00231 s<sup>-1</sup><br />
| <br />
|[https://2008.igem.org/Team:KULeuven/Model/Filter#References [3<html>]</html>]<br />
|-<br />
| d<sub>open mRNA T7 complex</sub><br />
| 0.00231 s<sup>-1</sup><br />
| <br />
|[https://2008.igem.org/Team:KULeuven/Model/Filter#References [3<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Equilibrium constants<br />
|-<br />
| K<sub>eq 1</sub><br />
| 0,015 [M]<br />
| between closed and open T7 mRNA, models competition, experimental<br />
| [https://2008.igem.org/Team:KULeuven/Model/Filter#References [1<html>]</html> [2<html>]</html>]<br />
|-<br />
| K<sub>eq 2</sub><br />
| 0.0212 [M]<br />
| between closed T7 mRNA and key unlocked mRNA complex, models competition, experimental<br />
| [https://2008.igem.org/Team:KULeuven/Model/Filter#References [1<html>]</html> [2<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Rate constants<br />
|-<br />
| k<sub>dis</sub><br />
| 0.00416 s<sup>-1</sup><br />
| estimate derived from experimental values<br />
| [https://2008.igem.org/Team:KULeuven/Model/Filter#References [1<html>]</html>]<br />
|-<br />
| k<sub>complex</sub><br />
| 0.00237 s<sup>-1</sup><br />
| estimate derived from experimental values<br />
| [https://2008.igem.org/Team:KULeuven/Model/Filter#References [1<html>]</html>]<br />
|-<br />
| k<sub>closed</sub><br />
| 500 s<sup>-1</sup><br />
| estimate derived from experimental values<br />
| [https://2008.igem.org/Team:KULeuven/Model/Filter#References [1<html>]</html>]<br />
|-<br />
| k<sub>open</sub><br />
| 7.5 s<sup>-1</sup><br />
| estimate derived from experimental values<br />
| [https://2008.igem.org/Team:KULeuven/Model/Filter#References [1<html>]</html>]<br />
|-<br />
| k<sub>translation</sub><br />
| 0.167 s<sup>-1</sup><br />
| estimate: lock defined translation rate for T7 RNA pol<br />
| [https://2008.igem.org/Team:KULeuven/Model/Filter#References [7<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Transcription rates<br />
|-<br />
| TetR_var_transcr_rate<br />
| p(TetR) dependent <br />
| (RiboKey) between 5E-5 and 0.0125 s<sup>-1</sup> ~ [aTc]<br />
| [https://2008.igem.org/Team:KULeuven/Model/Filter#References [6<html>]</html>]<br />
|-<br />
| k<sub>mRNA T7</sub><br />
| 0,0011 s<sup>-1</sup><br />
| weak constitutive promoter J23109<br />
| [https://2008.igem.org/Team:KULeuven/Model/Filter#References [7<html>]</html>]<br />
|}<br />
<br />
<b>Remark:</b> The key-lock system has been enhanced to 0.3%-14% (new parameters have been added)<br />
<br />
=== Models ===<br />
<br />
==== CellDesigner ([https://static.igem.org/mediawiki/2008/1/10/Filter_CellDesigner.zip SBML file])====<br />
<br />
[[Image:Filter_CellDesigner.png|800px|center|filter]]<br />
<br />
==== Matlab ([https://static.igem.org/mediawiki/2008/c/ce/Filter_Matlab.zip SBML file])====<br />
<br />
[[Image:Filter_Matlab.jpg|center]]<br />
<br />
=== Simulations ===<br />
<br />
[[Image:Sim_filter_1.png|800px|center|filter]]<br />
<br />
====1. AND gate of the filter====<br />
<br />
In the simulation we can clearly see this series of events:<br />
[[Image:Filter 1.PNG|right]]<br />
* when dark blue(ribokey) starts to increase, red (T7) also starts to increase, giving rise to an increasing amount of lactonase (blue) = AND-GATE.<br />
* when dark blue(ribokey) starts to decrease, red (T7) also starts to decrease, but much slower. The lactonase also starts to decrease, as it should be.<br />
The short lifetime of the ribokey compared to the lifetime of the T7-protein, guarantees that the AND-GATE always works perfectly fine: when there's no more input, the ribokey will rapidly decrease (and disappear) and makes sure that the AND-GATE is not activated anymore, even when the T7-protein is slowly decreasing.<br />
<br />
====2. Filtering in practice====<br />
<br />
In the simulation three kinds of inputpulses have been used:<br />
<br />
*First pulse: 300s<br />
**small peak of lactonase<br />
**no influence on the timer<br />
<br />
*Second pulse: 1000s<br />
**medium peak of lactonase<br />
**influences the timer by levelling the timing capabilities, but it doesn't reset the timer<br />
<br />
*Third pulse: 5000s<br />
**huge peak of lactonase<br />
**reset of the timer: amount of complex goes to zero<br />
<br />
====3. Proof of filtering capacities====<br />
<br />
As proof of the filtering capacities, we'll compare the maximum amount of GFP with the maximum amount of Lactonase as a function of the pulse length of TetR. We've chosen Lactonase and GFP because they differ only in the absence or presence of the filter, all other parameters are identical (eg. degradation rate). The model used here consists only of the filter, the output and the lactonase productions subsystems.<br />
<br />
The left figures shows us a clear difference between the amount of GFP and lactonase for almost every pulse length. The explanation for the (almost constant) big difference for pulses longer than 1000 seconds is the following. For such long pulses, the filter can be considered as fully active, such that the only differences between the output and the lactonase production are characteristics of the ribokey/lock and the difference in transcription rates between E. coli's RNA polymerase II and phage T7's polymerase. <br />
For a pulse shorter than 1000 seconds the filter is not fully active. This creates a filtering effect. If we just look at the first 1000 seconds (right figures), we see that the amount of Lactonase remains more or less the same while the amount of GFP increases very fast.<br />
<html><br />
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<div class="noborder" style="overflow: auto; width: 800px; height: 520px;"><br />
<div class="noborder" style="width: 1050px;"> <br />
<img src="https://static.igem.org/mediawiki/2008/1/18/Filter_amount_vs_pulse.png" style="float: left; width: 500px; height: 500px; margin: 0 5px;" /><br />
<img src="https://static.igem.org/mediawiki/2008/3/32/Filter_amount_vs_pulse_zoomed.png" style="float: left; width: 500px; height: 500px; margin: 0 5px;" /><br />
</div></div></div></html><br />
<br />
<br><br />
<br />
Another way to see the filtering effect is to look at the ratio of GFP to Lactonase. This figure clearly shows the disproportionality between GFP and lactonase for the pulses shorter than 1000 seconds. The best filtering effect occurs for pulses around 100 seconds. This figure also shows that placing the filter in front of a signal wil lower this signal with a factor of at least 25 (full throughput in the filter). This base factor should be multiplied by ~8 for pules around 100 seconds. This is the filtering effect quantitated: pulses of only 100 seconds will produce almost 25 * 8 = 200 times more GFP than Lactonase. A factor 25 characteristic of the system and a variable extra filtering factor that is a function of pulse length and is maximal (8) for pulses of 100 seconds.<br />
<br />
<html><br />
<div class="center"><br />
<div class="noborder" style="overflow: auto; width: 900px; height: 370px;"><br />
<div class="noborder" style="width: 850px;"> <br />
<img src="https://static.igem.org/mediawiki/2008/0/0c/GFP_Lactonase.png" style="float: left; width: 400px; height: 350px; margin: 0 5px;" /><br />
<img src="https://static.igem.org/mediawiki/2008/1/13/GFP_lactonase_norm.png" style="float: left; width: 400px; height: 350px; margin: 0 5px;" /><br />
</div></div></div></html><br />
<br />
<br><br />
<br />
Another nice way to visualazise the same filtering effect can be seen in the following figures. In the left figure, 4 input pulses are given in sequence. The first lasts 100 seconds, the second 500, the third 2500 while the last one remains indefinateley.<br />
The maximal output (result of the 4th pulse), both non-filtered and filtered are set to 1, normalizing the signals. It is now very easy to see the filtering in effect on the zoomed-in right figure and the ratio's can be compared with the y-values of the previous figure.<br />
<br />
<html><br />
<div class="center"><br />
<div class="noborder" style="overflow: auto; width: 900px; height: 320px;"><br />
<div class="noborder" style="width: 900px;"> <br />
<img src="https://static.igem.org/mediawiki/2008/1/18/Filter_extra.png" style="float: left; width: 440px; height: 300px; margin: 0 5px;" /><br />
<img src="https://static.igem.org/mediawiki/2008/0/0b/Filter_extra_zoom.png" style="float: left; width: 440px; height: 300px; margin: 0 5px;" /><br />
</div></div></div></html><br />
<br />
<br><br />
<br />
=== References ===<br />
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=utf-8"/><br />
<title>Bibliography</title><br />
</head><br />
<body><br />
<table style="border-collapse:collapse;line-height:1.1em;"><br />
<tr style="vertical-align:top;"><td>[1]</td><td style="padding-left:4pt;">“Berkeley2006-RiboregulatorsMain - IGEM”; http://parts2.mit.edu/wiki/index.php/Berkeley2006-RiboregulatorsMain.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[2]</td><td style="padding-left:4pt;">F.J. Isaacs et al., “Engineered riboregulators enable post-transcriptional control of gene expression,” <span style="font-style:italic;">Nat Biotech</span>, vol. 22, Jul. 2004, pp. 841-847. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1038/nbt986&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Engineered%20riboregulators%20enable%20post-transcriptional%20control%20of%20gene%20expression&amp;rft.jtitle=Nat%20Biotech&amp;rft.stitle=Nat%20Biotech&amp;rft.volume=22&amp;rft.issue=7&amp;rft.aufirst=Farren%20J&amp;rft.aulast=Isaacs&amp;rft.au=Farren%20J%20Isaacs&amp;rft.au=Daniel%20J%20Dwyer&amp;rft.au=Chunming%20Ding&amp;rft.au=Dmitri%20D%20Pervouchine&amp;rft.au=Charles%20R%20Cantor&amp;rft.au=James%20J%20Collins&amp;rft.date=2004-07&amp;rft.pages=841-847&amp;rft.issn=1087-0156"></span></td></tr><br />
<br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[3]</td><td style="padding-left:4pt;">J.A. Bernstein et al., “Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays,” <span style="font-style:italic;">Proceedings of the National Academy of Sciences of the United States of America</span>, vol. 99, Jul. 2002, pp. 9697–9702. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1073/pnas.112318199&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Global%20analysis%20of%20mRNA%20decay%20and%20abundance%20in%20Escherichia%20coli%20at%20single-gene%20resolution%20using%20two-color%20fluorescent%20DNA%20microarrays&amp;rft.jtitle=Proceedings%20of%20the%20National%20Academy%20of%20Sciences%20of%20the%20United%20States%20of%20America&amp;rft.stitle=Proc%20Natl%20Acad%20Sci%20U%20S%20A.%20&amp;rft.volume=99&amp;rft.issue=15&amp;rft.aufirst=Jonathan%20A.&amp;rft.aulast=Bernstein&amp;rft.au=Jonathan%20A.%20Bernstein&amp;rft.au=Arkady%20B.%20Khodursky&amp;rft.au=Pei-Hsun%20Lin&amp;rft.au=Sue%20Lin-Chao&amp;rft.au=Stanley%20N.%20Cohen&amp;rft.date=2002-07-23&amp;rft.pages=9697%E2%80%939702"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[4]</td><td style="padding-left:4pt;">“IGEM:Tsinghua/2007/Projects/RAP - OpenWetWare”; http://www.openwetware.org/wiki/IGEM:Tsinghua/2007/Projects/RAP#Model_and_simulation.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[5]</td><td style="padding-left:4pt;">M. Gonzalez et al., “Lon-mediated proteolysis of the Escherichia coli UmuD mutagenesis protein: in vitro degradation and identification of residues required for proteolysis,” <span style="font-style:italic;">Genes Dev.</span>, vol. 12, Dec. 1998, pp. 3889-3899. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1101/gad.12.24.3889&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Lon-mediated%20proteolysis%20of%20the%20Escherichia%20coli%20UmuD%20mutagenesis%20protein%3A%20in%20vitro%20degradation%20and%20identification%20of%20residues%20required%20for%20proteolysis&amp;rft.jtitle=Genes%20Dev.&amp;rft.volume=12&amp;rft.issue=24&amp;rft.aufirst=Martin&amp;rft.aulast=Gonzalez&amp;rft.au=Martin%20Gonzalez&amp;rft.au=Ekaterina%20G.%20Frank&amp;rft.au=Arthur%20S.%20Levine&amp;rft.au=Roger%20Woodgate&amp;rft.date=1998-12-15&amp;rft.pages=3889-3899"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<br />
<tr style="vertical-align:top;"><td>[6]</td><td style="padding-left:4pt;">M. Bon, S.J. McGowan, and P.R. Cook, “Many expressed genes in bacteria and yeast are transcribed only once per cell cycle,” <span style="font-style:italic;">FASEB J.</span>, vol. 20, Aug. 2006, pp. 1721-1723. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1096/fj.06-6087fje&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Many%20expressed%20genes%20in%20bacteria%20and%20yeast%20are%20transcribed%20only%20once%20per%20cell%20cycle&amp;rft.jtitle=FASEB%20J.&amp;rft.volume=20&amp;rft.issue=10&amp;rft.aufirst=Michael&amp;rft.aulast=Bon&amp;rft.au=Michael%20Bon&amp;rft.au=Simon%20J.%20McGowan&amp;rft.au=Peter%20R.%20Cook&amp;rft.date=2006-08-01&amp;rft.pages=1721-1723"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[7]</td><td style="padding-left:4pt;">“Part:BBa J23109 - partsregistry.org”; http://partsregistry.org/Part:BBa_J23109.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[8]</td><td style="padding-left:4pt;">J.P. McDonald et al., “Regulation of UmuD cleavage: role of the amino-terminal tail,” <span style="font-style:italic;">Journal of Molecular Biology</span>, vol. 282, Oct. 1998, pp. 721-730. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1006/jmbi.1998.2044&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Regulation%20of%20UmuD%20cleavage%3A%20role%20of%20the%20amino-terminal%20tail&amp;rft.jtitle=Journal%20of%20Molecular%20Biology&amp;rft.volume=282&amp;rft.issue=4&amp;rft.aufirst=John%20P&amp;rft.aulast=McDonald&amp;rft.au=John%20P%20McDonald&amp;rft.au=Erinn%20E%20Maury&amp;rft.au=Arthur%20S%20Levine&amp;rft.au=Roger%20Woodgate&amp;rft.date=1998-10-02&amp;rft.pages=721-730"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
</table></body><br />
<br />
</html></div>BNathaliehttp://2008.igem.org/Team:KULeuven/Ethics/Robotics/RoboticsTeam:KULeuven/Ethics/Robotics/Robotics2008-10-29T23:06:27Z<p>BNathalie: </p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
__NOTOC__<br />
== Biological robotics ==<br />
In more than one way, one easily recognizes some of the preliminary features of nanorobotics in Dr. Coli. Nanorobots are described as devices that can manipulate objects at the nanometer scale. This is the same order of magnitude as the size of atoms and molecules. Indeed, Dr. Coli is a kind of robot, that can deliver drugs where and when they are needed inside the human body. However, we would exaggerate saying it is a nanodevice. The typical dimensions of an ''E. coli'' cell are micrometers (10^-6) rather than nanometers (10^-9). This does, however, not stop us from treating Dr. Coli in terms of robotics.<br />
=== Three laws of robotics ===<br />
In his short story “Runaround”, published in 1942, the Russian writer (and professor in biochemistry!) Isaac Asimov introduced his “Three laws of Robotics”. They are:<br />
<blockquote>A robot may not injure a human being or, through inaction, allow a human being to come to harm.</blockquote><br />
<blockquote>A robot must obey orders given to it by human beings, except where such orders would conflict with the First Law.</blockquote><br />
<blockquote>A robot must protect its own existence as long as such protection does not conflict with the First or Second Law.</blockquote><br />
We will think about Dr. Coli here, using the “Three laws of Robotics” as a framework for further reflection.<br />
<br />
In “Do No Harm To Humans: Real-life Robots Obey Asimovs Laws”, the writers point to an interesting dilemma in working with robots. On the one side, it is easy to make robots do all kinds of stuff, thereby obeying the laws of robotics, if they are far away enough from mankind. On the other side, as long as robots work slow enough, they say, the robots cannot violate the laws. <br />
<blockquote>The trade-off between safety and performance is the name of the game in physical human-machine interactions. (6)</blockquote><br />
<br />
This understanding makes our reflection on Dr. Coli safety issues not easier. On the contrary, it makes it even more difficult. Dr. Coli does work the closest to man one can imagine: inside his body. Even more, it is practically impossible, and indeed even heavily unwanted, that the speed or efficiency of Dr. Coli is lowered.<br />
<br />
This second understanding imposes a lot of responsibility to the creators of Dr. Coli. We will have to go through the three laws of robotics and discuss in detail how we can make sure that Dr. Coli does not violate a single one of them. <br />
=== First Law ===<br />
<blockquote>A robot may not injure a human being or, through inaction, allow a human being to come to harm.</blockquote><br />
Asimov’s ideas about the three laws of robotics date from the first half of the twentieth century. In these times, no one was actually concerned about environmental health, global warming and fossil fuels. It is therefore no surprise that the first law of robotics needs critical review. We now are concerned about environmental health, global warming and fossil fuels (or the lack thereof). In this view, we can, and should in fact, expand the first law of robotics. Not only is a robot—or in our analysis, Dr. Coli—prohibited to allow a human being to come to harm, it should also be prohibited to allow any other part of nature to get harmed. Under any part of nature, we mean the environment in its broadest sense. It is indeed extremely important not to create such things, that can interfere with nature and, by doing so, harm it.<br />
<br />
This remark is a topic of general concern and is one of the issues that has been discussed maybe more than all other synthetic biology topics. It is in this context that a lot of discussion resembles the issues discussed during the Asilomar conference of 1975 on genetic engineering. During that conference, organized by Paul Berg, the world’s leading scientists heavily discussed the safety of recombinant technologies. That science was brand-new in those years and raised a lot of questions about whether it was safe to alter DNA. <br />
<br />
Much of the concerns resulted in regulations and agreements that are very common today (for instance biological and physical containment) or are either still actual for synthetic biology. Therefore, we will focus on Dr. Coli and reformulate the question concerning Dr. Coli and the First Law of robotics as following:<br />
<blockquote>How can we make sure that Dr. Coli does not harm any part of the enivronment?</blockquote><br />
We notice that we explicitly formulate the broad meaning of the word environment. This environment consists of three parts: the patient, other human beings and the non-human nature.<br />
==== Patient ====<br />
Taking the built-in safety mechanisms of Dr. Coli (see project description page) into account, the only way for him to bring harm to the patient is when he is given to a patient that is ill, but has not the illness the specific Dr. Coli is specialized in. Another possible danger is that Dr. Coli is given to or taken in by a non-infected patient. That way, Dr. Coli’s memory is in the 0-state when initially given to the patient and will not be activated, as there is no input signal. This is indeed a dangerous situation, for this situation will lead to uncontrolled cell growth that can only be stopped by using the right antibiotic treatment. This is to be avoided, as antibiotics are now, more than before, to be used with care to avoid the development of multiresistant strains. <br />
<br />
We can stress the importance of medical doctors here. Dr. Coli is indeed no different than other drugs in that it is dangerous to give the medicine to a healthy person or a person with a different problem. However, it is a very easy and irresponsible attitude to completely pass on the safety issues about Dr. Coli to the medical staff. We cannot hold them fully responsible for possible problems with Dr. Coli.<br />
==== Other human beings ====<br />
Dr. Coli is a bacterium that resides in the colon. Keeping in mind that the average household has not the same kind of safety regulation as the average biotechnological lab, we can be sure that Dr. Coli will be transmitted to people coming near the patient. This should, however, not pose a problem. In the case the patient is ill, Dr. Coli’s memory will be in a switched on state once it leaves the patient. This means that as long as no input signal is present, Dr. Coli will count to the moment he will kill himself.<br />
<br />
In the case the patient is not ill at all, or has a different illness, Dr. Coli can in fact spread in human beings, other than the patient. <br />
==== Non-human nature ====<br />
Also here, we can divide the risks into two classes. On the one hand, Dr. Coli is dangerous for the non-human nature after he has passed the intestinal system of the patient. On the other hand, Dr. Coli is dangerous for the non-human nature prior to administration to the patient. One can for instance spoil some Dr. Coli solution or a leak in growth reactors in the pharmacological factory can originate. However, such accidents can be monitored appropriately and heavy consequences can be prohibited by means of antiseptic agents.<br />
<br />
=== Second Law ===<br />
<blockquote>A robot must obey orders given to it by human beings, except where such orders would conflict with the First Law.</blockquote><br />
We can think about this law the following way. The orders given to Dr. Coli is to heal the patient when and where necessary. Furthermore, Dr. Coli is instructed to die after a period of absence of illness signal. This is encoded in its “software”, its genes. So the only way for Dr. Coli not to obey his orders is to mutate. There is no way of completely preventing this. Regular sequencings to check whether the sequence is still consistent and changing to a fresh culture derived from an early seed culture should become part of every production plan for Dr. Coli and become a standardized procedure. An other thing that can happen to Dr. Coli is the loss of the plasmid containing the working mechanism of Dr. Coli. This can, however, be more or less overcome by integrating the plasmid in the chromosome. <br />
=== Third Law ===<br />
<blockquote>A robot must protect its own existence as long as such protection does not conflict with the First or Second Law.</blockquote><br />
This means that, based on easy logic, we can alter the Third Law to the following statement:<br />
<blockquote>If the existence of a robot conflicts with the First or the Second Law, it may not defend its own existence.</blockquote><br />
A conflict with the first law can, for instance, occur when Dr. Coli escapes the biological and physical containment imposed on it. We will provide a more elaborate workout for the issues with Dr. Coli ending up in the environment. We quote the Summary Statement of the Asilomar Conference on Recombinant DNA Molecules:<br />
<blockquote>Containment of potentially biohazardous agents can be achieved in several ways. The most significant [...] is the use of biological barriers. These barriers are of two types: 1) fastidious bacterial hosts unable to survive in natural environments, and 2) nontransmissible and equally fastidious vectors [...] able to grow only in specified hosts. (7) </blockquote><br />
<br />
One of the best possibilities to put up a biological barrier, is replacing an essential gene by the Dr. Coli construct. A conflict with the second law is less feasible, as Dr. Coli does not have a choice but to follow his genes. Mutating or dying are the only things Dr. Coli can do to escape the directions given to him by us. As we said before, screening methods can monitor any abnormal behavior and easy and quick measures can be taken.<br />
=== Dr. Coli safety alterations ===<br />
It has not escaped our notice that if we ever want to implement Dr. Coli, more safety alterations have to be added. First of all is ''E. coli'' certainly not the best bacterium available to apply the system in human beings. <br />
<br />
One of the best bacteria to carry out the function of Dr. Coli, would be ''Lactobacillus''. ''Lactobacilli'' are gut-friendly gram-positive bacteria, widely used in probiotics. As ''Lactobacilli'' are already present in the gut, they are completely harmless. An important feature of ''Lactobacillus'' is the fact that it’s an immobile bacterium. So once arrived in the gut, it stays there. The risk of spreading and contamination of the environment is thus minimized.<br />
<br />
To reduce the risk that activated Dr. Coli can reside somewhere we don’t want it to be, we can place a second AND-gate at the beginning of our system. This double input can only activate the system if both signals are available. The signal of sickness is one input, the other input can be a substance we add to the diet of our patient. We have to make sure that the second input is very specific and is something normal people don’t take in. <br />
<br />
Another point we have to consider, is the fact that the second input has to be an essential substance for the cells. So if there’s only one input available, Dr. Coli dies. Up to now, Dr. Coli’s active elements are gathered on plasmids. This is, however, far from the best method for making cells for administration in the gastro-intestinal system, where they can make a therapeutical agent. An improved version of Dr. Coli should for instance contain the active elements in the chromosome.<br />
<br />
Researchers at the VIB (Flanders Institute for Biotechnology) already demonstrated a mechanism of this kind. They engineered a ''Lactobacillus'' strain to produce IL-10 in the treatment of Crohn’s disease. To be allowed to do studies in clinical phase with testing on humans (they already arrived at that point), they needed a perfectly safe mechanism that allows the ''Lactobacillus'' to live in the patient, but inhibits growth outside the body. The mechanism used by the VIB research group made use of a ''Lactobacillus'' strain in which the thymidylate synthase gene (thyA) has been replaced with a gene producing a pharmacological agent (in their case, IL-10). This means that the bacterium will die in response to thymine and thymidine starvation (Ahmad, S. L, Kirk, S. H. and Eisenstark, A. (1998) Thymine metabolism and thymineless death in prokaryotes and eukaryotes. Annu. Rev. Microbiol. 52,591-625.).<br />
<br />
Finally we want to emphasize that our Dr. Coli is a proof of principle and that the concept is far from ready to be implemented in human beings.</div>BNathaliehttp://2008.igem.org/Team:KULeuven/Tools/ComponentsTeam:KULeuven/Tools/Components2008-10-29T23:03:17Z<p>BNathalie: </p>
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<ul><br />
<li><a href="https://2008.igem.org/Team:KULeuven/Project/Input"><br />
<img src="https://static.igem.org/mediawiki/2008/a/a9/Pictogram_input.png">Input</a></li><br />
<li><a href="https://2008.igem.org/Team:KULeuven/Project/Output"><br />
<img src="https://static.igem.org/mediawiki/2008/d/db/Pictogram_output.png">Output</a></li><br />
<li><a href="https://2008.igem.org/Team:KULeuven/Project/Filter"><br />
<img src="https://static.igem.org/mediawiki/2008/e/ed/Pictogram_filter.png">Filter</a></li><br />
<li><a href="https://2008.igem.org/Team:KULeuven/Project/Inverter"><br />
<img src="https://static.igem.org/mediawiki/2008/e/e7/Pictogram_inverter.png">InverTimer</a></li><br />
<li><a href="https://2008.igem.org/Team:KULeuven/Project/Reset"><br />
<img src="https://static.igem.org/mediawiki/2008/e/ed/Pictogram_reset.png">Reset</a></li><br />
<li><a href="https://2008.igem.org/Team:KULeuven/Project/CellDeath"><br />
<img src="https://static.igem.org/mediawiki/2008/c/c6/Pictogram_celldeath.png">Cell Death</a></li><br />
<li class="last"><a href="https://2008.igem.org/Team:KULeuven/Project/Memory"><br />
<img src="https://static.igem.org/mediawiki/2008/c/c2/Pictogram_memory.png">Memory</a></li><br />
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</html></div>BNathaliehttp://2008.igem.org/Team:KULeuven/Project/InverterTeam:KULeuven/Project/Inverter2008-10-29T23:01:25Z<p>BNathalie: </p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Scripting}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
[[Image:pictogram_inverter.png|120px|right]]<br />
==Invertimer==<br />
<br />
===BioBricks===<br />
[[Image:project_inverter.jpg|center]]<br />
<br />
===Components===<br />
The input signal for the InverTimer is filtered by using the T7 RNA polymerase promoter ([http://partsregistry.org/Part:BBa_I712074 '''BBa_I712074''']) and RiboLock3d ([http://partsregistry.org/Part:BBa_J23078 '''BBa_J23078''']). The InverTimer's output signal is the LuxI protein, encoded by [http://partsregistry.org/Part:BBa_C061 '''BBa_C0061''']. The basis of the InverTimer system is the LacI repressor molecule, encoded by [http://partsregistry.org/Part:BBa_C0012 '''BBa_C0012'''], and the lac promotor ([http://partsregistry.org/Part:BBa_R0011 '''BBa_R0011''']).<br />
<br />
===Action===<br />
When there is a consistent input signal that can penetrate the [https://2008.igem.org/Team:KULeuven/Project/Filter Filter], LacI is produced from the T7 promoter with the adjacent RiboLock. This LacI ([http://partsregistry.org/Part:BBa_C0012 '''BBa_C0012''']) contains a C-terminal [https://2008.igem.org/Team:KULeuven/Literature#LVA_tag LVA tag] for faster degradation and thus better responsiveness. LacI inhibits transcription from the lac promoter just downstream, acting as an inverter for the output LuxI signal. When no (or not enough, because of the filter) signal is present, the LacI repressor will not be made and LuxI will be produced. It is thus clear that when the filter system produces an input signal for the inverter, there will be repression of LuxI production. Otherwise, when no input signal from the filter system is present, LuxI will be produced.<br />
<br />
[[Image:3OC6HSL.gif|right]]<br />
<br />
When LuxI is present, it functions as a timer, slowly producing the quorum-sensing molecule [http://partsregistry.org/3OC6HSL 3OC6HSL] from hexanoyl-ACP and SAM. 3OC6HSL has a very long lifetime if no quorum-quenching [https://2008.igem.org/Team:KULeuven/Project/Reset Lactonase] is present and will accumulate. Not only inside the cell but it will also diffuse into the medium. <br />
<br />
3OC6HSL is the bridge that links this [https://2008.igem.org/Team:KULeuven/Project/Inverter InverTimer] to the next device in our system, [https://2008.igem.org/Team:KULeuven/Project/CellDeath Cell Death]. Our [https://2008.igem.org/Team:KULeuven/Project/Reset Reset] device also impinges upon this small signaling molecule, making it a true crossroads in our total system.<br />
<br />
{{:Team:KULeuven/Tools/Components}}</div>BNathaliehttp://2008.igem.org/Team:KULeuven/Project/OutputTeam:KULeuven/Project/Output2008-10-29T22:57:29Z<p>BNathalie: </p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Scripting}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
[[Image:pictogram_output.png|120px|right]]<br />
<br />
==Output==<br />
<div style="float:right">[[Image:GFP.png|350px]]</div><br />
We have been searching for an easy accessible output system, that could provide us qualitative and quantitative information in a quick and easy way. The choice for GFP was easily made.<br />
<br />
===BioBricks===<br />
[[Image:project_output.jpg|left]]<br />
<br><br />
<br />
<br />
===Components===<br />
A GFP with a C-terminal [https://2008.igem.org/Team:KULeuven/Literature#LVA_tag LVA tag] for rapid degradation of the protein (Andersen et al, 1998) was chosen so that we can follow the output signal quickly in time. The Tet promoter ([http://partsregistry.org/Part:BBa_R0040 '''BBa_R0040''']) behaves as described in the [https://2008.igem.org/Team:KULeuven/Project/Input Input] section. For fine-tuning reasons, the RBS was chosen [http://partsregistry.org/Part:BBa_B0032 '''BBa_B0032'''] with a relative efficiency of 0,3.<br />
<br />
===Action===<br />
This is a very easy system. As the [https://2008.igem.org/Team:KULeuven/Project/Input Input] results in inactivation of the ''tet''R repressor, there is GFP production, proportional to the input signal.<br />
<br />
==References==<br />
Appl Environ Microbiol. 1998 Jun;64(6):2240-6.<br />
New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria.<br />
Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, Molin S.<br />
PMID: 9603842<br />
<br />
{{:Team:KULeuven/Tools/Components}}</div>BNathaliehttp://2008.igem.org/Team:KULeuven/Project/InputTeam:KULeuven/Project/Input2008-10-29T22:51:52Z<p>BNathalie: </p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Scripting}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
[[Image:pictogram_input.png|120px|right]]<br />
<br />
==Old Input==<br />
Searching for an input signal that was easily turned on and off, we chose to use the light-sensing device [http://partsregistry.org/Part:BBa_M30109 '''BBa_M30109''']. This device has already been used by a number of teams, but for the sake of completeness, we describe it here again. We also gave it our own (working) number: [http://partsregistry.org/Part:BBa_K145011 '''BBa_K145011'''].<br />
<br />
===BioBricks===<br />
[[Image:project_input.jpg|center]]<br />
<br />
===Components===<br />
The first step is to produce ''ho1'' (heme oxigenase 1) that converts heme to biliverdin IXalpha. This enzyme is encoded by [http://partsregistry.org/Part:BBa_I15008 '''BBa_I15008''']. The second step is to produce ''PcyA'' (phycocyanobilin:ferredoxin oxidoreductase) that converts the biliverdin IXalpha to phycocyanobilin. This enzyme is encoded by [http://partsregistry.org/Part:BBa_I15009 '''BBa_I15009''']. This phycocyanobilin associates with the light receptive domain Cph1 of a Cph1/EnvZ fusion protein, encoded by [http://partsregistry.org/Part:BBa_I15010 '''BBa_I15010'''].<br />
<br />
An extra need for the input mechanism is the presence of the TetR repressor. This molecule is encoded by part [http://partsregistry.org/Part:BBa_C0040 '''BBa_C0040'''], and contains an [https://2008.igem.org/Team:KULeuven/Literature#LVA_tag LVA tag] for rapid degradation.<br />
<br />
===Action===<br />
[[Image:input_1.jpg|160px|right]]<br />
The ''ho1'' and ''PcyA'' coding parts are placed under a Pbad promoter ([http://partsregistry.org/Part:BBa_I13453 '''BBa_I13453''']). As the AraC repressor is present in the cell, phycocyanobilin will only be made upon activation with arabinose. The coding part for the Cph1/EnvZ fusion protein is placed under control of a pTet promoter ([http://partsregistry.org/Part:BBa_R0040 '''BBa_R0040''']). This means that, as TetR ([http://partsregistry.org/Part:BBa_C0040 '''BBa_C0040''']), the Tet repressor, is present, the fusion protein will only be produced when aTc is added to the medium. This gives us the possibility to test the system with an exogenous memory instead of the endogenous one, which will then be added later on in the project (see [https://2008.igem.org/Team:KULeuven/Project/Memory here]).<br />
<br />
[[Image:input.png|left|350px]]<br />
The Cph1 part of the Cph1/EnvZ fusion protein acts as the light sensor, the EnvZ part is a kinase. Upon radiation with 660nm (red) light, the fusion protein gets dephosphorylated. This renders the kinase in an inactive state. The active kinase phosphorylates the molecule OmpR. A high concentration of the phosphorylated ompR represses the OmpF promoter ([http://partsregistry.org/Part:BBa_R0084 '''BBa_R0084''']). A lower concentration activates it. So when 660nm light is irradiated on the input mechanism, the concentration of phosphorylated OmpR drops and the OmpF promoter is turned on.<br />
<br />
==New Input==<br />
Doing some more searching around and after a couple of e-mails, we realised that [http://partsregistry.org/Part:BBa_M30109 '''BBa_M30109'''] caused a lot of problems for a lot of teams in 2007. That is why we decided to a more reliable, althought practically more difficult system.<br />
<br />
===BioBricks===<br />
[[Image:project_input_bis.jpg|left]] [[Image:tetrepressor.jpg|right|Tet repressor binding to the Tet promoter|290px]]<br />
<br><br />
<br />
<br />
===Components===<br />
[http://partsregistry.org/Part:BBa_J23116 '''BBa_J23116'''] is a constitutive promotor, member of the '''BBa_J23xxx''' family promotors. The [http://partsregistry.org/Part:BBa_B0032 '''BBa_B0032'''] has an efficiency of 0,3 and [http://partsregistry.org/Part:BBa_B0015 '''BBa_B0015'''] is an efficient terminator. [http://partsregistry.org/Part:BBa_C0040 '''BBa_C0040'''] is a protein coding sequence that codes for the ''tet''T repressor (see Figure). The [http://partsregistry.org/Part:BBa_R0040 '''BBa_R0040'''] promotors of Output, Memory and Filter are turned off by the constitutively produced ''tet''R repressor protein. By adding anhydrotetracycline (aTc), the repressor protein can be disengaged from the operator sequence and the Output, Memory and Filter will be turned on.<br />
<br />
{{:Team:KULeuven/Tools/Components}}</div>BNathaliehttp://2008.igem.org/Team:KULeuven/Model/OutputTeam:KULeuven/Model/Output2008-10-29T22:47:41Z<p>BNathalie: /* Position in the system */</p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Scripting}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
[[Image:pictogram_output.png|120px|right]]<br />
<br />
== Output ==<br />
<br />
=== Position in the system ===<br />
<br />
The output system is one of three modules directly linked to the input. The output system is a simple gene regulation, of which transcription is repressed by TetR - and can be activated by anhydrotetracyclin. The output signal is GFP (green fluorescent protein).<br />
<br />
In the applied project of dealing with Crohn's disease this would be replaced by the drug necessary to cure the local inflammation. The drug concentration would be proportional to the amount of local inflammation sensed.<br />
<br />
=== Describing the system ===<br />
see also: [https://2008.igem.org/Team:KULeuven/Project/Output Project:Output]<br />
<br />
[[Image:Output_BioBrick.jpg|center]]<br />
<br />
==== ODE's ====<br />
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==== Parameters ====<br />
<br />
{| width=80% style="border: 1px solid #003E81; background-color: #EEFFFF;"<br />
|+ ''Parameter values (Output)''<br />
|-<br />
! width=15% | Name<br />
! width=15% | Value<br />
! width=40% | Comments<br />
! width=10% | Reference<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Degradation Rates<br />
|-<br />
| d<sub>GFP</sub><br />
| d<sub>LVA</sub> = 2.814E-4 s<sup>-1</sup><br />
| LVA-tag reduces lifetime to 40 minutes<br />
| [https://2008.igem.org/Team:KULeuven/Model/Output#References [1<html>]</html>] [https://2008.igem.org/Team:KULeuven/Model/Output#References [4<html>]</html>]<br />
|-<br />
| d<sub>mRNA_GFP</sub><br />
| 0.0023 s<sup>-1</sup><br />
| <br />
| [https://2008.igem.org/Team:KULeuven/Model/Output#References [2<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Transcription Rates<br />
|-<br />
| TetR_var_transcr_rate<br />
| p(TetR) dependent <br />
| (GFP) between 5E-5 and 0.0125 s<sup>-1</sup> ~ [aTc]<br />
| [https://2008.igem.org/Team:KULeuven/Model/Output#References [3<html>]</html>]<br />
|-<br />
! colspan="4" style="border-bottom: 1px solid #003E81;" | Translation Rates<br />
|-<br />
| k<sub>GFP</sub><br />
| 0.167 s<sup>-1</sup><br />
| translation rate for B0032 RBS (0.3 relative efficiency)<br />
| [https://2008.igem.org/Team:KULeuven/Model/Output#References [5<html>]</html>]<br />
|}<br />
<br />
=== Models ===<br />
==== CellDesigner ([https://static.igem.org/mediawiki/2008/5/53/Output_CellDesigner.zip SBML file]) ====<br />
<br />
[[Image:Output_CellDesigner.png|500px|center|Output]]<br />
<br />
<br />
==== Matlab ([https://static.igem.org/mediawiki/2008/1/16/Output_Matlab.zip SBML file]) ====<br />
<br />
<br />
[[Image:Output_Matlab.jpg|center]]<br />
<br />
<br />
=== Simulations ===<br />
<br />
Several simulations were conducted. The following two show the transient respons and eventual saturation of the output system to a low (left figure) and a high (right figure) input signal (TetR = 5E-5 vs 0.0125 s<sup>-1</sup>). There is a great distinction in the number of GFP molecules between the two input signals. All graphs have amounts (number of molecules in the cell) plotted vs time, measured in seconds.<br />
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<br />
The next figure shows the switching behaviour of the output system to an light signal turning on and off:<br />
<br />
[[Image:Sim_output_3.png|450px|center]]<br />
<br />
=== Sensitivity Analysis ===<br />
<br />
[[Image:Sens output2.png|center]]<br />
<br />
=== References ===<br />
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</head><br />
<body><br />
<table style="border-collapse:collapse;line-height:1.1em;"><br />
<tr style="vertical-align:top;"><td>[1]</td><td style="padding-left:4pt;">“ETHZ/Parameters - IGEM07”; https://2007.igem.org/ETHZ/Parameters.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[2]</td><td style="padding-left:4pt;">J.A. Bernstein et al., “Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays,” <span style="font-style:italic;">Proceedings of the National Academy of Sciences of the United States of America</span>, vol. 99, Jul. 2002, pp. 9697–9702. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1073/pnas.112318199&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Global%20analysis%20of%20mRNA%20decay%20and%20abundance%20in%20Escherichia%20coli%20at%20single-gene%20resolution%20using%20two-color%20fluorescent%20DNA%20microarrays&amp;rft.jtitle=Proceedings%20of%20the%20National%20Academy%20of%20Sciences%20of%20the%20United%20States%20of%20America&amp;rft.stitle=Proc%20Natl%20Acad%20Sci%20U%20S%20A.%20&amp;rft.volume=99&amp;rft.issue=15&amp;rft.aufirst=Jonathan%20A.&amp;rft.aulast=Bernstein&amp;rft.au=Jonathan%20A.%20Bernstein&amp;rft.au=Arkady%20B.%20Khodursky&amp;rft.au=Pei-Hsun%20Lin&amp;rft.au=Sue%20Lin-Chao&amp;rft.au=Stanley%20N.%20Cohen&amp;rft.date=2002-07-23&amp;rft.pages=9697%E2%80%939702"></span></td></tr><br />
<br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[3]</td><td style="padding-left:4pt;">M. Bon, S.J. McGowan, and P.R. Cook, “Many expressed genes in bacteria and yeast are transcribed only once per cell cycle,” <span style="font-style:italic;">FASEB J.</span>, vol. 20, Aug. 2006, pp. 1721-1723. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1096/fj.06-6087fje&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Many%20expressed%20genes%20in%20bacteria%20and%20yeast%20are%20transcribed%20only%20once%20per%20cell%20cycle&amp;rft.jtitle=FASEB%20J.&amp;rft.volume=20&amp;rft.issue=10&amp;rft.aufirst=Michael&amp;rft.aulast=Bon&amp;rft.au=Michael%20Bon&amp;rft.au=Simon%20J.%20McGowan&amp;rft.au=Peter%20R.%20Cook&amp;rft.date=2006-08-01&amp;rft.pages=1721-1723"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[4]</td><td style="padding-left:4pt;">J.B. Andersen et al., “New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria,” <span style="font-style:italic;">Applied and Environmental Microbiology</span>, vol. 64, Jun. 1998, pp. 2240–2246. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=New%20Unstable%20Variants%20of%20Green%20Fluorescent%20Protein%20for%20Studies%20of%20Transient%20Gene%20Expression%20in%20Bacteria&amp;rft.jtitle=Applied%20and%20Environmental%20Microbiology&amp;rft.stitle=Appl%20Environ%20Microbiol.%20&amp;rft.volume=64&amp;rft.issue=6&amp;rft.aufirst=Jens%20Bo&amp;rft.aulast=Andersen&amp;rft.au=Jens%20Bo%20Andersen&amp;rft.au=Claus%20Sternberg&amp;rft.au=Lars%20Kongsbak%20Poulsen&amp;rft.au=Sara%20Petersen%20Bj%C3%B8rn&amp;rft.au=Michael%20Givskov&amp;rft.au=S%C3%B8ren%20Molin&amp;rft.date=1998-06&amp;rft.pages=2240%E2%80%932246"></span></td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<tr style="vertical-align:top;"><td>[5]</td><td style="padding-left:4pt;">“Part:BBa B0032 - partsregistry.org”; http://partsregistry.org/Part:BBa_B0032.</td></tr><br />
<tr><td colspan="2">&nbsp;</td></tr><br />
<br />
</table></body><br />
</html></div>BNathaliehttp://2008.igem.org/File:K145279.JPGFile:K145279.JPG2008-10-29T22:44:43Z<p>BNathalie: uploaded a new version of "Image:K145279.JPG"</p>
<hr />
<div></div>BNathaliehttp://2008.igem.org/Team:KULeuven/LigationTeam:KULeuven/Ligation2008-10-29T22:41:27Z<p>BNathalie: /* New Devices */</p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Scripting}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
== New Parts ==<br />
<br />
{| style="background:#99CCFF; color:black; width:85%; text-align:center;" border="1" cellpadding="2" cellspacing="0"<br />
! width=75|BioBrick<br />
! width=645|Description<br />
! width=70|Status<br />
! width=70|Works?<br />
|- style="background:#ffffff; color:black; text-align:center;"<br />
| [http://partsregistry.org/Part:BBa_K145001 K145001] || T7 DNA polymerase (no tag)|| [[Image:correct.JPG|center|30px]] || <br />
|- style="background:#ffffff; color:black; text-align:center;"<br />
| [http://partsregistry.org/Part:BBa_K145013 K145013] || antisense LuxI|| [[Image:correct.JPG|center|30px]] || <br />
|- style="background:#ffffff; color:black; text-align:center;"<br />
| [http://partsregistry.org/Part:BBa_K145014 K145014] || T7 DNA polymerase with UmuD tag|| [[Image:correct.JPG|center|30px]] || <br />
|- style="background:#ffffff; color:black; text-align:center;"<br />
| [http://partsregistry.org/Part:BBa_K145015 K145015] || GFP with LVA tag|| [[Image:correct.JPG|center|30px]] || [[Image:correct.JPG|center|30px]]<br />
|- style="background:#ffffff; color:black; text-align:center;"<br />
| [http://partsregistry.org/Part:BBa_K145150 K145150] || hybrid promoter|| [[Image:correct.JPG|center|30px]] || <br />
|- style="background:#ffffff; color:black; text-align:center;"<br />
| [http://partsregistry.org/Part:BBa_K145151 K145151] || ccdB coding region || [[Image:correct.JPG|center|30px]] || <br />
|}<br />
<br />
==New Devices ==<br />
<br />
{| style="background:#99CCFF; color:black; width:85%; text-align:center;" border="1" cellpadding="2" cellspacing="0"<br />
! width=100 |'''BioBrick''' <br />
! width=450 | '''Components'''<br />
! width=200 | '''Description'''<br />
! width=100 | '''Status*'''<br />
! width=100 | '''Works?'''<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145201 K145201] || [[Image:K145201.JPG|center]] || Input || [[Image:correct.JPG|center|30px]] || [[Image:correct.JPG|center|30px]]<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145205 K145205] || [[Image:K145205.JPG|center]] || Output (LVA tag)|| [[Image:correct.JPG|center|30px]] || [[Image:correct.JPG|center|30px]]<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145252 K145252] || [[Image:K145252.JPG|center]] || Output (no tag) || [[Image:correct.JPG|center|30px]] || [[Image:correct.JPG|center|30px]]<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145280 K145280] || [[Image:K145280.JPG|center]]|| Input-Output: test || [[Image:correct.JPG|center|30px]] || [[Image:correct.JPG|center|30px]]<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145279 K145279] || [[Image:K145279.JPG|center]] || Input-Output: test || [[Image:correct.JPG|center|30px]] || [[Image:correct.JPG|center|30px]]<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145258 K145258] || [[Image:K145258.JPG|center]] || Memory - part 1 || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145259 K145259] || [[Image:K145259.JPG|center]] || Memory - part 2 || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145210 K145210] || [[Image:K145210.JPG|center]] || Memory || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145211 K145211] || [[Image:K145211.JPG|center]] || Memory - part 3 || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145215 K145215] || [[Image:K145215.JPG|center|150px]] || Filter - part 1 || [[Image:correct.JPG|center|30px]] || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145278 K145278] || [[Image:K145278.JPG|center]] || Filter (old) - part 2 || ||<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145277 K145277] || [[Image:K145277.JPG|center]] || Filter (new) - part 2 || ||<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145275 K145275] || [[Image:K145275.JPG|center]] || Filter (old) || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145276 K145276] || [[Image:K145276.JPG|center]] || Filter (new) || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145253 K145253] || [[Image:K145253.JPG|center]] || Invertimer (old) - part 1 || PCR || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145255 K145255] || [[Image:K145255.JPG|center]] || Invertimer (new) - part 1 || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145254 K145254] || [[Image:K145254.JPG|center]] || Invertimer - part 2 || [[Image:correct.JPG|center|30px]] || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145220 K145220] || [[Image:K145220.JPG|center|300px]] || Invertimer (new) || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145104 K145104] || [[Image:K145104.JPG|center]] || Invertimer (old) || PCR || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145251 K145251] || [[Image:K145251.JPG|center]] || Reset (old) || PCR || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145225 K145225] || [[Image:K145225.JPG|center]] || Reset (new) || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145256 K145256] || [[Image:K145256.JPG|center]] || Cell Death - part 1 || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145257 K145257] || [[Image:K145257.JPG|center]] || Cell Death - part 2 || [[Image:correct.JPG|center|30px]] ||<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145230 K145230] || [[Image:K145230.JPG|center]] || Cell Death || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145273 K145273] || [[Image:K145273.JPG|center]] || test K145150 - part 2 || [[Image:correct.JPG|center|30px]] || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145270 K145270] || [[Image:K145270.JPG|center]] || test K145150 - part 3 || ||<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145269 K145269] || [[Image:K145269.JPG|center]] || test K145150 - part 4 || [[Image:correct.JPG|center|30px]] || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145268 K145268] || [[Image:K145268.JPG|center]] || test K145150 - part 5 || ||<br />
|- style="background:#ffffff; color:black; text-align:center; height:45px"<br />
| [http://partsregistry.org/Part:BBa_K145267 K145267] || [[Image:K145267.JPG|center]] || test K145150 - part 6 || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145266 K145266] || [[Image:K145266.JPG|center|450px]] || test K145150|| || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145271 K145271] || [[Image:K145271.JPG|center]] || test filter (new) - part 3 || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145272 K145272] || [[Image:K145272.JPG|center]] || test filter (old) - part 3 || PCR || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145265 K145265] || [[Image:K145265.JPG|center|410px]] || test filter (old) || ||<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145264 K145264] || [[Image:K145264.JPG|center|410px]] || test filter (new) || ||<br />
|}</div>BNathaliehttp://2008.igem.org/Team:KULeuven/LigationTeam:KULeuven/Ligation2008-10-29T22:40:15Z<p>BNathalie: /* New Parts */</p>
<hr />
<div>{{:Team:KULeuven/Tools/Styling}}<br />
{{:Team:KULeuven/Tools/Scripting}}<br />
{{:Team:KULeuven/Tools/Header}}<br />
<br />
== New Parts ==<br />
<br />
{| style="background:#99CCFF; color:black; width:85%; text-align:center;" border="1" cellpadding="2" cellspacing="0"<br />
! width=75|BioBrick<br />
! width=645|Description<br />
! width=70|Status<br />
! width=70|Works?<br />
|- style="background:#ffffff; color:black; text-align:center;"<br />
| [http://partsregistry.org/Part:BBa_K145001 K145001] || T7 DNA polymerase (no tag)|| [[Image:correct.JPG|center|30px]] || <br />
|- style="background:#ffffff; color:black; text-align:center;"<br />
| [http://partsregistry.org/Part:BBa_K145013 K145013] || antisense LuxI|| [[Image:correct.JPG|center|30px]] || <br />
|- style="background:#ffffff; color:black; text-align:center;"<br />
| [http://partsregistry.org/Part:BBa_K145014 K145014] || T7 DNA polymerase with UmuD tag|| [[Image:correct.JPG|center|30px]] || <br />
|- style="background:#ffffff; color:black; text-align:center;"<br />
| [http://partsregistry.org/Part:BBa_K145015 K145015] || GFP with LVA tag|| [[Image:correct.JPG|center|30px]] || [[Image:correct.JPG|center|30px]]<br />
|- style="background:#ffffff; color:black; text-align:center;"<br />
| [http://partsregistry.org/Part:BBa_K145150 K145150] || hybrid promoter|| [[Image:correct.JPG|center|30px]] || <br />
|- style="background:#ffffff; color:black; text-align:center;"<br />
| [http://partsregistry.org/Part:BBa_K145151 K145151] || ccdB coding region || [[Image:correct.JPG|center|30px]] || <br />
|}<br />
<br />
==New Devices ==<br />
<br />
{| style="background:#99CCFF; color:black; width:85%; text-align:center;" border="1" cellpadding="2" cellspacing="0"<br />
! width=100 |'''BioBrick''' <br />
! width=450 | '''Components'''<br />
! width=200 | '''Description'''<br />
! width=100 | '''Status*'''<br />
! width=100 | '''Works?'''<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145201 K145201] || [[Image:K145201.JPG|center]] || Input || [[Image:correct.JPG|center|30px]] || [[Image:correct.JPG|center|30px]]<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145205 K145205] || [[Image:K145205.JPG|center]] || Output (LVA tag)|| [[Image:correct.JPG|center|30px]] || [[Image:correct.JPG|center|30px]]<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145252 K145252] || [[Image:K145252.JPG|center]] || Output (no tag) || [[Image:correct.JPG|center|30px]] || [[Image:correct.JPG|center|30px]]<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145280 K145280] || [[Image:K145280.JPG|center]]|| Input-Output: test || [[Image:correct.JPG|center|30px]] || [[Image:correct.JPG|center|30px]]<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145279 K145279] || [[Image:K145279.JPG|center]] || Input-Output: test || [[Image:correct.JPG|center|30px]] || [[Image:correct.JPG|center|30px]]<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145258 K145258] || [[Image:K145258.JPG|center]] || Memory - part 1 || LM|| <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145259 K145259] || [[Image:K145259.JPG|center]] || Memory - part 2 || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145210 K145210] || [[Image:K145210.JPG|center]] || Memory || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145211 K145211] || [[Image:K145211.JPG|center]] || Memory - part 3 || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145215 K145215] || [[Image:K145215.JPG|center|150px]] || Filter - part 1 || [[Image:correct.JPG|center|30px]] || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145278 K145278] || [[Image:K145278.JPG|center]] || Filter (old) - part 2 || ||<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145277 K145277] || [[Image:K145277.JPG|center]] || Filter (new) - part 2 || ||<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145275 K145275] || [[Image:K145275.JPG|center]] || Filter (old) || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145276 K145276] || [[Image:K145276.JPG|center]] || Filter (new) || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145253 K145253] || [[Image:K145253.JPG|center]] || Invertimer (old) - part 1 || PCR || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145255 K145255] || [[Image:K145255.JPG|center]] || Invertimer (new) - part 1 || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145254 K145254] || [[Image:K145254.JPG|center]] || Invertimer - part 2 || [[Image:correct.JPG|center|30px]] || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145220 K145220] || [[Image:K145220.JPG|center|300px]] || Invertimer (new) || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145104 K145104] || [[Image:K145104.JPG|center]] || Invertimer (old) || PCR || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145251 K145251] || [[Image:K145251.JPG|center]] || Reset (old) || PCR || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145225 K145225] || [[Image:K145225.JPG|center]] || Reset (new) || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145256 K145256] || [[Image:K145256.JPG|center]] || Cell Death - part 1 || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145257 K145257] || [[Image:K145257.JPG|center]] || Cell Death - part 2 || [[Image:correct.JPG|center|30px]] ||<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145230 K145230] || [[Image:K145230.JPG|center]] || Cell Death || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145273 K145273] || [[Image:K145273.JPG|center]] || test K145150 - part 2 || [[Image:correct.JPG|center|30px]] || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145270 K145270] || [[Image:K145270.JPG|center]] || test K145150 - part 3 || ||<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145269 K145269] || [[Image:K145269.JPG|center]] || test K145150 - part 4 || [[Image:correct.JPG|center|30px]] || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145268 K145268] || [[Image:K145268.JPG|center]] || test K145150 - part 5 || ||<br />
|- style="background:#ffffff; color:black; text-align:center; height:45px"<br />
| [http://partsregistry.org/Part:BBa_K145267 K145267] || [[Image:K145267.JPG|center]] || test K145150 - part 6 || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145266 K145266] || [[Image:K145266.JPG|center|450px]] || test K145150|| || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145271 K145271] || [[Image:K145271.JPG|center]] || test filter (new) - part 3 || || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145272 K145272] || [[Image:K145272.JPG|center]] || test filter (old) - part 3 || PCR || <br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145265 K145265] || [[Image:K145265.JPG|center|410px]] || test filter (old) || ||<br />
|- style="background:#ffffff; color:black; text-align:center; height:50px"<br />
| [http://partsregistry.org/Part:BBa_K145264 K145264] || [[Image:K145264.JPG|center|410px]] || test filter (new) || ||<br />
|}<br />
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<nowiki>* EP = just been electroporated, LM = Ligation mix has been made</nowiki></div>BNathaliehttp://2008.igem.org/Team:KULeuven/25_October_2008Team:KULeuven/25 October 20082008-10-29T22:38:24Z<p>BNathalie: New page: {{:Team:KULeuven/Tools/Styling}} {{:Team:KULeuven/Tools/Header}} {{:Team:KULeuven/Tools/New_Day/Date_Retriever}} == Miscellaneous == Poster & presentation</p>
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== Miscellaneous ==<br />
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Poster & presentation</div>BNathaliehttp://2008.igem.org/Team:KULeuven/15_October_2008Team:KULeuven/15 October 20082008-10-29T22:38:03Z<p>BNathalie: </p>
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== Miscellaneous ==<br />
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* Poster & presentation<br />
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* Electroporated the ligations of Monday 13, October ([http://partsregistry.org/Part:BBa_K145253 K145253], [http://partsregistry.org/Part:BBa_K145104 K145104], [http://partsregistry.org/Part:BBa_K145272 K145272] and [http://partsregistry.org/Part:BBa_K145251 K145251]). Hopefully we'll have colonies tomorrow.</div>BNathaliehttp://2008.igem.org/Team:KULeuven/15_October_2008Team:KULeuven/15 October 20082008-10-29T22:37:38Z<p>BNathalie: New page: {{:Team:KULeuven/Tools/Styling}} {{:Team:KULeuven/Tools/Header}} {{:Team:KULeuven/Tools/New_Day/Date_Retriever}} == Miscellaneaous == * Poster & presentation * Electroporated the ligat...</p>
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== Miscellaneaous ==<br />
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* Poster & presentation<br />
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* Electroporated the ligations of Monday 13, October ([http://partsregistry.org/Part:BBa_K145253 K145253], [http://partsregistry.org/Part:BBa_K145104 K145104], [http://partsregistry.org/Part:BBa_K145272 K145272] and [http://partsregistry.org/Part:BBa_K145251 K145251]). Hopefully we'll have colonies tomorrow.</div>BNathaliehttp://2008.igem.org/Team:KULeuven/9_November_2008Team:KULeuven/9 November 20082008-10-29T22:33:51Z<p>BNathalie: New page: {{:Team:KULeuven/Tools/Styling}} {{:Team:KULeuven/Tools/Header}} {{:Team:KULeuven/Tools/New_Day/Date_Retriever}} '''!! JAMBOREE !!''' Check our blog [http://igemkuleuven.wordpress.com...</p>
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'''!! JAMBOREE !!''' Check our blog [http://igemkuleuven.wordpress.com/ here]</div>BNathaliehttp://2008.igem.org/Team:KULeuven/8_November_2008Team:KULeuven/8 November 20082008-10-29T22:33:39Z<p>BNathalie: </p>
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'''!! JAMBOREE !!''' Check our blog [http://igemkuleuven.wordpress.com/ here]</div>BNathaliehttp://2008.igem.org/Team:KULeuven/8_November_2008Team:KULeuven/8 November 20082008-10-29T22:33:18Z<p>BNathalie: </p>
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'''!! JAMBOREE !!''' Check our blog [http://igemkuleuven.wordpress.com/ here]</div>BNathaliehttp://2008.igem.org/Team:KULeuven/8_November_2008Team:KULeuven/8 November 20082008-10-29T22:32:10Z<p>BNathalie: New page: '''!! JAMBOREE !!''' Check our blog [http://igemkuleuven.wordpress.com/ here]</p>
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<div> '''!! JAMBOREE !!''' Check our blog [http://igemkuleuven.wordpress.com/ here]</div>BNathaliehttp://2008.igem.org/Team:KULeuven/29_October_2008Team:KULeuven/29 October 20082008-10-29T22:29:47Z<p>BNathalie: New page: {{:Team:KULeuven/Tools/Styling}} {{:Team:KULeuven/Tools/Header}} {{:Team:KULeuven/Tools/New_Day/Date_Retriever}} == Miscellaneous == * Poster & presentation (as usual) * Finishing the ...</p>
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== Miscellaneous ==<br />
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* Poster & presentation (as usual)<br />
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* Finishing the wiki (adding information on the Data Analysis page, update Notebook, check for mistakes,...)<br />
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'''!! WIKI FREEZE !!'''<br />
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Notebook stops here</div>BNathaliehttp://2008.igem.org/Team:KULeuven/28_October_2008Team:KULeuven/28 October 20082008-10-29T22:27:07Z<p>BNathalie: New page: {{:Team:KULeuven/Tools/Styling}} {{:Team:KULeuven/Tools/Header}} {{:Team:KULeuven/Tools/New_Day/Date_Retriever}} == Miscellaneous == * We gave the presentation another go (in front of o...</p>
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== Miscellaneous ==<br />
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* We gave the presentation another go (in front of our advisors). There are still a few things that have to be corrected or improved, but we're getting beter.<br />
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* Included information about different potential test modules on the Data Analysis page.</div>BNathaliehttp://2008.igem.org/Team:KULeuven/27_October_2008Team:KULeuven/27 October 20082008-10-29T22:24:13Z<p>BNathalie: New page: {{:Team:KULeuven/Tools/Styling}} {{:Team:KULeuven/Tools/Header}} {{:Team:KULeuven/Tools/New_Day/Date_Retriever}} == Miscellaneous == * Poster & presentation (& rehearsal) * Data analys...</p>
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== Miscellaneous ==<br />
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* Poster & presentation (& rehearsal)<br />
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* Data analysis page is updated with information about the Output ([https://2008.igem.org/Team:KULeuven/Data/Output link]).</div>BNathaliehttp://2008.igem.org/Team:KULeuven/26_October_2008Team:KULeuven/26 October 20082008-10-29T22:22:10Z<p>BNathalie: New page: {{:Team:KULeuven/Tools/Styling}} {{:Team:KULeuven/Tools/Header}} {{:Team:KULeuven/Tools/New_Day/Date_Retriever}} == Miscellaneous == * Poster & presentation (& rehearsal) * Started che...</p>
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== Miscellaneous ==<br />
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* Poster & presentation (& rehearsal)<br />
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* Started checking the wiki.</div>BNathaliehttp://2008.igem.org/Team:KULeuven/24_October_2008Team:KULeuven/24 October 20082008-10-29T22:21:13Z<p>BNathalie: New page: {{:Team:KULeuven/Tools/Styling}} {{:Team:KULeuven/Tools/Header}} {{:Team:KULeuven/Tools/New_Day/Date_Retriever}} == Miscellaneous == Poster & presentation (& rehearsal)</p>
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== Miscellaneous ==<br />
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Poster & presentation (& rehearsal)</div>BNathaliehttp://2008.igem.org/Team:KULeuven/23_October_2008Team:KULeuven/23 October 20082008-10-29T22:18:41Z<p>BNathalie: New page: {{:Team:KULeuven/Tools/Styling}} {{:Team:KULeuven/Tools/Header}} {{:Team:KULeuven/Tools/New_Day/Date_Retriever}} == Miscellaneous == * Poster & presentation * The parts that were send ...</p>
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== Miscellaneous ==<br />
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* Poster & presentation<br />
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* The parts that were send to iGEM HQ, are now indicated as 'favorite parts'.</div>BNathalie