Template:Team:UC Berkeley/Notebook/BX notes

Sixpi 19:26, 18 September 2008 (UTC)
I made t_holin.term.(Pcon).T_antiholin.term - 4 variants with PconB5, PconC5, PconG6, and Ptet. I'll try attaching T_lysozyme to the front sometime soon.

Sixpi 21:26, 6 September 2008 (UTC)
I haven't updated this recently since classes began, but I'm working with Christie to try to finish more variations of the lysis device. We're still having problems with the T4 system, but we realized it was due to the transfers being incorrect. I hope to successfully assemble T_lysozyme.T_holin next Monday.

Sixpi 03:06, 21 August 2008 (UTC)
Wednesday: I picked colonies for the assemblies I did yesterday, except bxa153 did not grow, so I'll try again and gel purifying the bands.

I also amplified bxa80L and bxa80R, as well transforming the bxa77 plasmid I got from Jin's T4 antiholin strain into lefty so I can use it in 1-2-3 assembly.

Tuesday: I helped Sherine attempt an ELISA, as it was her last day and she wanted to try the experiment that was her final goal. The ELISA didn't work, maybe because we didn't induce with arabinose for a long enough period of time.

I also redid my lysis experiment, and I got positive results, so now I have 3 variations of the lysis device (Pbad.lambda lysozyme.lambda holin.terminator.{Pcon-B5,Pcon-C5,Pcon-G6}.lambda antiholin.terminator).

I transformed 6 assemblies today: bxa59,60, 61, 95, 96, and 153.

Monday: Attempted lysis experiment for Pbad.bxa50, 51, and 52 by diluting saturated cultures 100-fold in 5 mL of media, growing to mid-log(~3 hours), then splitting the cultures in half and inducing one half with 5 uL of 20% arabinose. However, I didn't actually manage to add arabinose since I took up arabinose with a pipet, then put the tip directly into the media, and then the arabinose went up the tip instead of down into the media.

Sunday: Picked colonies for Pbad.bxa50, 51, and 52.

Sixpi 20:13, 16 August 2008 (UTC)
Saturday: TODO: -assemble Pbad.bxa50, 51, and 52

Friday: Miniprepped 50,51,52,54. Digested with EcoRV/XhoI and BstAPI/XhoI, but didn't run gel yet.

Thursday: Picked colonies for bxa50, 51, 52, and 54.

Sixpi 17:33, 13 August 2008 (UTC)
Wednesday: TODO: -miniprep -digestion map -assemble Assemblies done today: bxa50, bxa51, bxa52, bxa54.

Sixpi 16:58, 12 August 2008 (UTC)
Tuesday: TODO: -pick colonies for assemblies/transfers done yesterday -miniprep cultures -digestion minipreps -send sequencing

Sixpi 16:40, 11 August 2008 (UTC)
Monday: TODO: -either transfer spvR or do modified 1-2-3 assembly for bxa54. -pick more colonies for bxa153 -send bxa50 that digestion mapped correctly for sequencing.

Sunday: Finished the modified 1-2-3 assemblies that were started yesterday. Sequencing results show that Pspv2 has a point mutation near the end, and my transfer for spvR did not actually work. bxa153 also did not assemble correctly.

Saturday: Miniprepped and digestion mapped the cultures picked yesterday, most of them turned out correct. The digestion map of bxa50 with AvrII and EcoRV was incorrect, so I'll miniprep the remaining cells I have left sometime. Did four 1-2-3 assemblies with the correctly mapped bxa6 and bxa7, went up to gel purification with six modified 1-2-3 assemblies.

Sixpi 16:35, 8 August 2008 (UTC)
Friday: TODO: -Digestion map pBad.bxa50 using AvrII and EcoRV -Miniprep colonies picked yesterday -Pick colonies for assemblies/transfers done yesterday -Do troublesome assemblies using modified 1-2-3 method pBad.bxa50 did not lyse in response to arabinose, so now I need to digestion map the part to see if it looks correct.

Sixpi 18:09, 7 August 2008 (UTC)
Thursday: TODO: -Dilute pBad.bxa50 100-fold, grow up to midlog, and try lysing with arabinose. -Pick colonies for transformations yesterday. -Analyze sequencing data -Try more assemblies if possible

Sixpi 02:01, 7 August 2008 (UTC)
Wednesday: UCSF bbq...a lot of running around, and also getting kicked in the shin once >.<

I set up 4 transfers and 2 assemblies. 3 of the transfers are retransfers of the different Pcon's, since the assemblies using those parts haven't been working. The last transfers is a redo of bxa22, since that seems to actually be Pcon-B5. The two assemblies are two new layer 3 assemblies that became possible when sequencing confirmed bxa78.

Sixpi 02:48, 6 August 2008 (UTC)
Tuesday: Today was the museum trip. I came in the morning to send off some samples for sequencing. When I got back in the afternoon, I had sequencing data for the samples I sent off on Saturday. Out of the 17 I sent off, only 3 worked. I need to figure out what to do about the ones that sequenced badly(mixed template and other strange things).

Sixpi 17:53, 4 August 2008 (UTC)
Monday: TODO: -Pick colonies for pBad.bxa50 -Analyze sequencing data -Figure out which transfers/assemblies still need to be redone so I can do more of layer 3.

Sunday: Finished miniprepping cultures picked Friday night, which included bxa50 (a lysis device minus the inducible promoter at the beginning). Didn't wait for sequencing data, but put pBad in front of two bxa50 samples that digestion mapped correctly.

Saturday: Started miniprepping cultures. Digestion mapped the cultures I miniprepped and sent off for sequencing.

Friday: Friend's birthday, came in the morning to pick some colonies, came back at night to pick more colonies.

Thursday:

Wednesday: Actually started new assemblies, made competent cells

Sixpi 16:14, 29 July 2008 (UTC)
Tuesday: TODO: -miniprep assemblies picked yesterday -analyze sequencing data for samples sent yesterday -finish updating sequencing log -make competent cells with the antiholin strain -start new assemblies

Sixpi 19:33, 28 July 2008 (UTC)
Monday: TODO: -pick colonies for assemblies done yesterday -miniprep cultures grown yesterday -analyze sequencing data -update sequencing log -plan ahead for rest of week

Sixpi 17:44, 27 July 2008 (UTC)
Sunday: TODO: -pick colonies for assemblies that turned out cotransformed, transfers -analyze sequencing data -possibly start more assemblies

Sixpi 16:37, 26 July 2008 (UTC)
Saturday: TODO: -miniprep bxa149 -analyze sequencing data -pick colonies for bxa11 and bxa129

Sixpi 16:35, 25 July 2008 (UTC)
Friday: TODO: -miniprep all blocks of culture picked yesterday -start more assemblies -send out miniprepped DNA for sequencing -amplify what we think is K112011 and K112201 on the stock plate so we can sequence/digestion map them -pick colonies for transfers done yesterday Didn't start assemblies, transfered the wrong thing for bxa11 yesterday, bxa129 is the same basic part but in a different vector, so retransferred that too. Sent out miniprepped DNA from assemblies out for sequencing.

Sixpi 15:38, 24 July 2008 (UTC)
Thursday: TODO: -pick colonies for both morning and afternoon assemblies -miniprep bxa9 grown up in righty, K112118 -minimeeting -analyze sequencing data -do 2 more transfers -start more assemblies

Didn't actually get to start more assemblies today, sequencing data showed I actually made <xis!.terminator, so now I have to go find the rbs.antiholin part I made.

Sixpi 02:25, 24 July 2008 (UTC)
Wednesday: Finished my assembly from yesterday, got back sequences for the assemblies I miniprepped yesterday - 3 out of 4 were correct. I miniprepped some stored bacteria for the one that was wrong, and then finished one more layer 1 assembly and 10 layer 2 assemblies.

Tuesday: Miniprepped the 2 blocks of culture I picked yesterday, digestion mapped, and did the digestion for the 1-2-3 assembly protocol for 5 of my layer 1 assemblies.

Sixpi 17:22, 21 July 2008 (UTC)
Monday: TODO: -miniprep colonies picked yesterday -digestion map miniprepped DNA -pick colonies from transfers/assemblies yesterday -if possible, start more assembly reactions

Sixpi 00:58, 21 July 2008 (UTC)
Sunday: bxa11 and bxa129 did not grow, so I redid those transfers, along with 6 others that did not work. I also started four assemblies.

Saturday: Miniprepped samples that Molly picked for me yesterday. Digestion mapping looked good for all but one of them, so I will have DNA to do assembly with tomorrow.

Friday: Miniprepped samples that I picked yesterday, and also ran digestion maps on them. About half of them looked ok, so

Thursday: Miniprepped colonies picked yesterday, picked colonies for transfers started yesterday.

Wednesday: Picked colonies for my transfers yesterday, started another set of transfers for which I didn't have DNA for yesterday.

Tuesday: I made my assembly tree and started transfers, also amplified the DNA I didn't have enough of.

Sixpi 17:04, 15 July 2008 (UTC)
Monday: I reattempted the Qiagen protocol, this time being more careful, but the result was the same. I then tried the Agencourt miniprep again and got better results. However, at this point we decided to give up on the assembly line method of creating composite parts and picked out around 75 parts we definitely want to make, and split those up among ourselves. I will be making the self lysis device - I've designed my assembly tree and I'll start making parts tomorrow.

Sunday: Started the last batch of 50 transfers for things we didn't have DNA for before and things that I just forgot to put on our list. Also attempted the magnetic miniprep using Qiagen's protocol, but that didn't work either - the genomic DNA stuck to the beads and the beads clumped together. Since we had no way of miniprepping the samples we grew up in culture yesterday, we just spun them down, discarded the media and put the blocks in the fridge.

Saturday: Kayaking! All the Berkeley bioengineering summer interns were invited to kayak at Pier 40 in SF. Our trip was approximately one hour, and after the kayaking session I went to jtown to look for origami paper, which I found.

Anyways, I went back to lab after the trip to attempt a miniprep in the 96 well format. Jin said I did all the steps correctly, but the DNA concentration was incredibly low. We're not sure what to try next....but we regrew cultures of the first set of transfers we did on 7/3 anyways.

Sixpi 00:13, 13 July 2008 (UTC)
Friday: Went ahead and attempted 9 assemblies, even though we don't have most of the DNA we should have and what we have is mostly unvalidated. Attempted with 2 uL each of lefty and righty plasmid, to see if the reaction will work with the DNA amount reduced. Also ran several digestion maps of various samples of DNA, but saw no bands for any of them. Therefore, we need to find a better way to miniprep hundreds of samples at once.

Thursday: Started miniprepping all the colonies we picked yesterday using columns in the morning, and finished 79. During our minimeeting, Chris said that the possibility of tube switches was too great, so we did not continue in the afternoon, and instead repicked the colonies again, this time into 96 well blocks to facilitate the use of magnetic beads.

Wednesday: Repicked all the colonies from the 7/3 set of transfers again, as the last miniprep did not work. Decided to pick in to 24 well blocks, since we want a larger volume of bacteria so we can obtain more DNA. Used 3 mL of appropriate 2 antibiotic 2YT media.

Tuesday: Spent most of the day reorganizing our spreadsheets, and began getting ready to run digestion mapping on all the things that Dirk miniprepped yesterday.

Sixpi 22:39, 7 July 2008 (UTC)
Monday: Redid transfers that failed again yesterday. Poured more plates, minipreped cultures that were picked yesterday. Running desperately low on pipet tips.

Sunday: Redid transfers that had failed the first time around. Picked new colonies from the first set of transfers that worked.

Sixpi 23:33, 5 July 2008 (UTC)
Saturday: Came in to find out that a large number of our transfers gave colonies, which meant Jin and Christie set up 200 colony PCRs yesterday! All but 4 of our basic part amplifications grew, so I minipreped the cultures we have so we can hopefully finish off the rest of the transfers tomorrow.

Friday: July 4th, did not come in.

Thursday: All 48 transfers from Wednesday failed. We think it might be because we dried the magnetic beads for too long during the purification step. Since time is of the essence, we started the rest of the transfers anyways, which amounted to 2 96 well plates worth. We also started to redo some of the failed transfers, but we have run out of DNA from some of our basic parts, so we also set up transformations to amplify the small amount of DNA we have left.

Wednesday: Reduced tree generator inefficiency by breaking down our list of parts into 3 sets, and running the program on each set separately. We will probably have some overlapping parts between the sets, but this can't be easily avoided without rewriting the entire program. We started 48 transfers as a small scale test before going up to full scale assembly.

Tuesday: Tests of our assembly vectors gave positive results, so we can begin transfers now. However, Jin still hasn't finished finalizing the list of composite parts we wish to attempt to make, so we did not actually begin today. Also, the assembly tree generator program that we have seems to give us inefficient trees, and the inefficiency grows as the size of the input grows, so we need to figure out some solution to this problem.

Sixpi 16:54, 1 July 2008 (UTC)
Monday: Since there were no colonies on the plates from yesterday, the vectors were not validated and we couldn't start the assembly process today. I set up another batch of tests for the vectors, this time with the real inserts that we will want in the respective vectors, which will give us K112202 in AC, K112118 in CA, K112704 in AK, K112109 in KA, K112709 in CK, and K112202 in KC.

Sunday: I asked Madhvi to move my plates down to the fridge for me. She didn't see any colonies.

Saturday: I didn't actually get to set up my transformations yesterday since we made time to plan out the next few weeks, so I came in today to test the vectors I finished preparing yesterday. I used some old dud inserts that had point mutations or deletions: K112311_1 in AC, K112313 in CA, K112316 in AK, K112311 in KA, K112201 in CK, and K112204 in KC.

Sixpi 21:10, 28 June 2008 (UTC)
Friday: Colony PCR of transformation from Thursday confirmed that the protocols were working, although the first batch of vectors was too dilute, and there were few colonies. The vector I prepared today were run on a traditional agarose gel and the bands were cut out, and I eluted with 50 uL of water in the final step of the zymo cleanup. Hopefully this will make the second batch more concentrated and the transformations I set up today work better.

We also discussed the upcoming assembly process, and who will do what job. It looks like we'll all be very busy for the next 2 or 3 weeks.

Sixpi 17:13, 27 June 2008 (UTC)
Thursday: Got sequences for my last part back (K112013), sequences were correct, so I am done with my basic parts. PCR for assembly vectors worked beautifully, retried ligating dud basic parts into assembly vectors and transforming.

Wednesday: Minimeeting, set up transformations for testing the assembly vectors. Set up PCR to make more assembly vectors, as first batch did not turn out very well.

Tuesday: Picked colonies for transformation yesterday.

Monday: Got sequencing data back, which was wrong yet again, so I restarted K112013 from PCR, and finished the transformation.

Sixpi 22:46, 22 June 2008 (UTC)
Sunday: Finished minipreps for Aron and Molly, also picked colonies for Madhvi. Reorganized E. coli plates, and restructured DNA stock database.

Saturday: Day off. Madhvi did miniprep for me.

Friday: Sequencing data came back, was scrambled, figured out that columns A and H, B and G, C and F, D and E got swapped, and was able to analyze results. Got a bad read again for K112013, so picked another colony and hoped it worked, otherwise I'll have to start over from PCR.

Thursday: Finished miniprep for two colonies from yesterday, sent DNA off for sequencing. Adobe Illustrator tutorial today.

Sixpi 22:45, 18 June 2008 (UTC)
Wednesday: Made more digested pBca1256 vector. Sequencing data has come in for K112000, K112001,K112002, K112004, K112013, and K112015. 4 of the 6 were perfect reads, but 2 were not, so I repicked colonies for those two parts, and will do a miniprep tomorrow.

Tuesday: Miniprep on the colonies I picked yesterday using the Qiagen miniprep kit.

Monday: Picked second colonies from the plates that had bad sequencing data, and grew them up in 3 ml of 2xYT media.

Sixpi 04:46, 16 June 2008 (UTC)
Sunday: I finished my DNA sequence analysis:

K112000~K112017 6.15.2008 Sequence Analysis

BX001:K112000 - Sequencing data totally off. BX002:K112001 - One possible point mutation at bp 685, T that should be a G, but chromatogram data is low, so unsure. The mutation is significant, as it changes a Gly to a stop codon. BX003:K112002 - Parent vector with mutations. BX004:K112003 - Perfect match. BX005:K112004 - One possible point mutation at bp 451, changing a Tyr to a Ser. Also, read data did not cover entire part. BX006:K112005 - Perfect match. BX007:K112006 - Perfect match. BX008:K112007 - Perfect match. BX009:K112008 - Perfect match. BX010:K112009 - Perfect match. BX011:K112010 - Perfect match. BX012:K112011 - Perfect match. BX013:K112012 - Perfect match. BX014:K112013 - Low read quality. BX015:K112014 - Perfect match. BX016:K112015 - Point mutation at bp 103, changing a Ile to a Thr. BX017:K112016 - Perfect match. BX018:K112017 - Perfect match.

Saturday: I came in to do a miniprep of the bacteria I picked yesterday using the Agencourt miniprep kit. This kit uses magnetic beads that bind dna to purify dna. The dna was sent out for sequencing analysis.

Friday: I spent a few hours waiting for our colonies to grow big enouogh to pick. When the colonies were ready, we made 2xYT media, and grew the colonies in 1 ml of media in a 96 well plate.

Sixpi 06:35, 13 June 2008 (UTC)
We had our first mini-meeting today, where we talked about what we have done for the last two weeks(somewhat), and about various subjects related to synthetic biology(more so). The meeting was interesting and not painful.

After the meeting, I went down to the lab to take out our PCR product from the machine. I then purified the product using clonewell gels, digested, cleaned up the reaction with the zymo kit, and the started the ligation. I then had to leave, so Molly will finish the transformation for me. In return, I'll do her mini-preps on Saturday.

Sixpi 22:04, 11 June 2008 (UTC)
Our oligos arrived today(yay!). We started making our basic parts. I made a mistake while making the master mix - I didn't know the dNTPs were at 10 mM instead of 2 mM, so I added 5 times as much dNTPs than needed. This means our PCR reaction has a good chance of failing, since dNTPs bind Mg, which the DNA polymerase needs in order to copy DNA. The extra dNTPs will probably outcompete the polymerase. No DNA was seen after running the eGel, so we set up the PCR reaction again, this time adding the correct amount of dNTPs.

Yesterday I poured more plates; 1 liter of Amp/Kan, 1 liter of Amp/Cam, and 1 liter of Cam/Kan.

Sixpi 23:31, 9 June 2008 (UTC)
Today I poured plates. There were 3 liters of LB/agar media, to which I added Spe. The 3 liters made 150 plates.

Sixpi 00:02, 5 June 2008 (UTC)
In our demos this morning we transformed bacteria, plated them, picked colonies, grew them up, and extracted DNA. The afternoon was taken up by designing oligos for the basic parts will start making next week(hopefully).

Sixpi 05:12, 4 June 2008 (UTC)
Today we continued our part making demo. Yesterday we set up the PCR reaction, and today we cleaned up the PCR, set up the digestion, and also ran an analytical gel of the PCR product.