Team:KULeuven/15 July 2008

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Lab Work

Wet lab

Today was our first day in the lab. We prepared LB-medium, ampicillin,... and Stefanie was very excited about pouring plates! Everything is now ready for the serious labwork: from tips to cleaning our benches. Today was also a historic day, because our big scheme was born (see Parts-page). We also made a first attempt to punch out a part from the registry. We started with the input device: [http://partsregistry.org/Part:BBa_M30109 M30109]. Then we transformed competent cells (DH5alpha) with this part.

Dry lab

General

We discovered that the LVA tagging of T7 RNA polymerase is a no go, as the C-terminus is part of the active site of the enzyme. We'll have to do N-terminal tagging. This should be able to work, see the crystal structure of T7. We could use the Lon-mediated degradation of lambda excisionase (Xis) and E.coli UmuD gene product. A 30-40 amino terminal AA of UmuD should do the trick. So we will have to construct a T7 polymerase with UmuD tag to make it degrade faster. For more information on this tag, check out Literature.

Modeling

Parameters, parameters, parameters, ... and Matlab, aah! (total smiley face)

We discovered that the ETHZ 2007 parameter page reveals information on certain degradation and kinetical constants. The link is supplied on the Modeling page.

Meeting

Present: All students, IT, KM, JW, JR, BDM, AC

Some remarks concerning our project:

They showed us where we could find the right E.coli strains.
There could be a problem with photobleaching of eCFP.
The production of eCFP could also be lowered by the low efficiency of the RBS.
Will there be enough ribokey? (Hanne says: ENHANCER!).
Will there be enough lactonase?
Maybe we should put tetR under control of his own promotor and lift it out of the cell death construct.
We can inhibit our system exogenously by adding lactonase to our medium.
We should use different ori's for our different plasmids, perhaps we'll have to link some devices.
The engineers should make a simple scheme that indicates which parameters are important for the sensitivity of our system.

Remarks

How great it is to have a sandbox!

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 == Lab Work ==
-=== Wetlab ===
+=== Wet lab ===
+Today was our first day in the lab. We prepared LB-medium, ampicillin,... and Stefanie was very excited about pouring plates! Everything is now ready for the serious labwork: from tips to cleaning our benches. Today was also a historic day, because our big scheme was born (see [https://2008.igem.org/Team:KULeuven/Parts Parts]-page). We also made a first attempt to punch out a part from the registry. We started with the input device: [http://partsregistry.org/Part:BBa_M30109 M30109]. Then we transformed competent cells (DH5alpha) with this part.
 === Dry lab ===
-Discovered that the LVA tagging T7 RNA polymerase is a no go as the C-terminus is part of the active site of the enzyme. We'll have to do N-terminal tagging, this could work, see the crystal structure of T7. See the link under literature on this wiki. Lon-mediated degradation of lambda excisionase (Xis) and E.coli UmuD gene product. 30-40 amino temrinal AA of UmuD should do the trick.
+==== General ====
+We discovered that the LVA tagging of T7 RNA polymerase is a no go, as the C-terminus is part of the active site of the enzyme. We'll have to do N-terminal tagging. This should be able to work, see the crystal structure of T7. We could use the Lon-mediated degradation of lambda excisionase (Xis) and E.coli UmuD gene product. A 30-40 amino terminal AA of UmuD should do the trick. So we will have to construct a T7 polymerase with UmuD tag to make it degrade faster. For more information on this tag, check out [https://2008.igem.org/Team:KULeuven/Literature Literature].
-== Modeling ==
+==== Modeling ====
-Parameters, parameters, parameters, ...
+Parameters, parameters, parameters, ... and Matlab, aah! (total smiley face)
+We discovered that the ''ETHZ 2007 parameter page'' reveals information on certain degradation and kinetical constants. The link is supplied on the Modeling page.
+== Meeting ==
+Present: All students, IT, KM, JW, JR, BDM, AC
+Some remarks concerning our project:
+* They showed us where we could find the right E.coli strains.
+* There could be a problem with photobleaching of eCFP.
+* The production of eCFP could also be lowered by the low efficiency of the RBS.
+* Will there be enough ribokey? (Hanne says: ENHANCER!).
+* Will there be enough lactonase?
+* Maybe we should put tetR under control of his own promotor and lift it out of the cell death construct.
+* We can inhibit our system exogenously by adding lactonase to our medium.
+* We should use different ori's for our different plasmids, perhaps we'll have to link some devices.
+* The engineers should make a simple scheme that indicates which parameters are important for the sensitivity of our system.
 == Remarks ==
-Project was checked by the presentation audience
+How great it is to have a sandbox!