Difference between revisions of "Davidson Projects with Updates"

Revision as of 21:43, 23 June 2008

Building LuxI sender TESTER cell

R0011 (verified) + F1610 (not right part from registry)
Going after S03608 (pLac_RBS_LuxI) from 2008 paper registry :-(
If we get this and verify, we are done with this. If not, we are in need of LuxI part.

Building LuxR receiver TESTER cell

Part K09100 has been built.
Ready to test.

Building LasI sender TESTER cell

R0011+ S03154.

Building LasR receiver TESTER cell

Ligatiion 1A S03156 + B0015
Ligation 1B R0079 + E0240
Ligation 1C B0015 + R0079

Ligation 2: Successful Ligation 1A + uccessful Ligation 1B
Ligation 3: Successful Ligation 2 + R0011

Testing Crosstalk between Lux and Las

1) Mix LuxI sender cells with LasR receiver tester cells: negative control
2) Mix LasI sender cells with LuxR receiver tester cells: negative control
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green
5) Verify LuxR receiver tester cells don't glow solo
6) Verify LasR receiver tester cells don't glow solo

AND gate using pTetLac promoter

We have successfully built the pTetLac AND promoter: K091101.
This is sequence verified.
Now we need the various LacI proteins (I12, X86 and I12+X86 variants).
We need tetR repressor protein.
We need to see if this AND gate works as designed.
We need to test output to see if the two halves are balanced.

Building XOR Gate

List of auto-inducers and their catalog numbers.

The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone (3OC6HSL)}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented by Egland and Greenberg

Oligos_to_Build: Sequences we will need to make this XOR gate.

Testing XOR Gate

We will need to have constitutive LasR and LuxR made in these cells.
Then we will need to hit them with the two chemicals AHL types and commercial sources.