Team:KULeuven/6 August 2008

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Lab Work

Wet Lab

The electrocompetent cells were tested with pUC and we had a lot of colonies so these are working great. Ligation products were transformed to these electrocompetent cells ([http://partsregistry.org/Part:BBa_E0022 E0022]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_J23109 J23109]+[http://partsregistry.org/Part:BBa_J23032 J23032], [http://partsregistry.org/Part:BBa_I712074 I712074]+[http://partsregistry.org/Part:BBa_J23032 J23032], [http://partsregistry.org/Part:BBa_K145015 K145015]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_K145015 K145015]+[http://partsregistry.org/Part:pSB1A2 pSB1A2], [http://partsregistry.org/Part:BBa_C0062 C0062]+[http://partsregistry.org/Part:BBa_B0015 B0015] and pUC as a control).

We miniprepped the colonies of the ligations that succeeded so fat. We also made digest of [http://partsregistry.org/Part:BBa_J23032 J23032], [http://partsregistry.org/Part:BBa_F1610 F1610], [http://partsregistry.org/Part:BBa_J23022 J23022] and [http://partsregistry.org/Part:BBa_B0015 B0015]. A picture of the result can be found below.

The three punched parts of the memory gave a few colonies. One of them did not give any colonies at all. The colonies we had were plated on new plates.

Picture of the gel containing the digests: J23032, J23022 and B0015

Dry Lab

General

Check possibility of hybrid promoter to connect memory and timer (LuxI). This is to replace the antisense LuxI. Promotor would look something like this:
Operator_{HSL_LuxR} --- -35 box --- Operator_{CII P22} --- -10 box

Hybrid promoter has been made! [http://partsregistry.org/Part:BBa_K145150 Hooray for BBa_K145150!] It should be activated by HSL-LuxR and repressed by P22 C2 (which is produced when the memory is OFF, hereby repressing LuxI synthesis) Check tomorrow how to best obtain the sequence.

Literature
[http://jb.asm.org/cgi/content/full/190/13/4392?view=long&pmid=18083819 Lux regulatory region (pLux)]

[http://www.jbc.org/cgi/content/full/272/3/1646/f1 P22 C2 repressor operators]

PS: P22 C2 is homologous to lambda CI while P22 C1 is homologous to lambda CII

Modeling

The antisense part has been remodeled to more accurately show its leakiness. This however appears not to be a problem at all. Plus we can always replace it with the hybrid promoter.

Started work on the sensitivity analyses. So far so good, as it would appear.

Further work on the memory because we got some new parameters. The switching has become somewhat trickier now.

New modeling of the antisense LuxI and its target LuxI mRNA shows that this leaky repression is not a problem for the system. Still looking at the composite promoter though.

Wiki

Dr. Coli is everywhere, even at the URL.

Remarks

Discovery of the day. Two people beat us to the punch: [http://www.diagnosticinformationsystem.com/bios.html 1] and [http://www.sscofcny.com/doctors/coli.htm 2]

Sindsdien hangt uw haar slap - Maarten

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+{{:Team:KULeuven/Tools/Header}}
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 === Wet Lab ===
-*The electrocompetent cells were tested with pUC and we had a lot of colonies so these are working great. *Ligation products were transformed to these electrocompetent cells (E0022+B0015, J23109+J23032, I712074+J23032, GFP+B0015, GFP+pSB1A2, C0062+B0015, pUC as a control).
+The electrocompetent cells were tested with pUC and we had a lot of colonies so these are working great. Ligation products were transformed to these electrocompetent cells ([http://partsregistry.org/Part:BBa_E0022 E0022]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_J23109 J23109]+[http://partsregistry.org/Part:BBa_J23032 J23032], [http://partsregistry.org/Part:BBa_I712074 I712074]+[http://partsregistry.org/Part:BBa_J23032 J23032], [http://partsregistry.org/Part:BBa_K145015 K145015]+[http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_K145015 K145015]+[http://partsregistry.org/Part:pSB1A2 pSB1A2], [http://partsregistry.org/Part:BBa_C0062 C0062]+[http://partsregistry.org/Part:BBa_B0015 B0015] and pUC as a control).
-*We made digest of J23032, F1610, J23022, B0015.
-*Miniprep colonies of the ligation.
-*The three punched parts of the memory gave a few colonies. One of them did not give any colonies at all. The colonies we had were plated on new plates.
-=== Dry Lab ===
+We miniprepped the colonies of the ligations that succeeded so fat. We also made digest of [http://partsregistry.org/Part:BBa_J23032 J23032], [http://partsregistry.org/Part:BBa_F1610 F1610], [http://partsregistry.org/Part:BBa_J23022 J23022] and [http://partsregistry.org/Part:BBa_B0015 B0015]. A picture of the result can be found below.
-Check possibility of hybrid promotor to connect memory and timer (LuxI). This is to replace the antisense LuxI. Promotor would look something like this: <br>
+The three punched parts of the memory gave a few colonies. One of them did not give any colonies at all. The colonies we had were plated on new plates.
-Operator<sub>HSL_LuxR</sub> '''---''' -35 box '''---''' Operator<sub>CII P22</sub> '''---''' -10 box
-New modeling of the antisense LuxI and its target LuxI mRNA shows that this leaky repression is not a problem for the system. Still looking at the composite promoter though.
+[[image:gel-06-8.jpg|center|thumb|500px|Picture of the gel containing the digests:  J23032, J23022 and B0015]]
+=== Dry Lab ===
+==== General ====
+Check possibility of hybrid promoter to connect memory and timer (LuxI). This is to replace the antisense LuxI. Promotor would look something like this: <br>
+Operator<sub>HSL_LuxR</sub> '''---''' -35 box '''---''' Operator<sub>CII P22</sub> '''---''' -10 box
+Hybrid promoter has been made! [http://partsregistry.org/Part:BBa_K145150 Hooray for BBa_K145150!] It should be activated by HSL-LuxR and repressed by P22 C2 (which is produced when the memory is OFF, hereby repressing LuxI synthesis) Check tomorrow how to best obtain the sequence.
 '''Literature''' <br>
 [http://jb.asm.org/cgi/content/full/190/13/4392?view=long&pmid=18083819 Lux regulatory region (pLux)]
-== Modeling ==
+[http://www.jbc.org/cgi/content/full/272/3/1646/f1 P22 C2 repressor operators]
-== Wiki ==
+PS: P22 C2 is homologous to lambda CI while P22 C1 is homologous to lambda CII
+==== Modeling ====
+The antisense part has been remodeled to more accurately show its leakiness. This however appears not to be a problem at all. Plus we can always replace it with the hybrid promoter.
+Started work on the sensitivity analyses. So far so good, as it would appear.
+Further work on the memory because we got some new parameters. The switching has become somewhat trickier now.
+New modeling of the antisense LuxI and its target LuxI mRNA shows that this leaky repression is not a problem for the system. Still looking at the composite promoter though.
+==== Wiki ====
 Dr. Coli is everywhere, even at the URL.
@@ Line 28: / Line 45: @@
 == Remarks ==
-Discovery of the day. Two people beat us to the punch: [http://www.diagnosticinformationsystem.com/bios.html 1] and [http://www.sscofcny.com/doctors/coli.htm 2]
+* Discovery of the day. Two people beat us to the punch: [http://www.diagnosticinformationsystem.com/bios.html 1] and [http://www.sscofcny.com/doctors/coli.htm 2]
-Sindsdien hangt uw haar slap -Maarten
+* Sindsdien hangt uw haar slap - Maarten