Team:Goettingen/week10-3

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Team Goettingen

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#3 Chemoreceptor Library - 10th Week

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V07_02

Startpoint of Saturated mutagenesis experiment!

Experiment:
We aim to apply directed evolution to reprogram the bacterial chemoreceptor TAR in order to enable the perception of novel substances. A saturated mutagenesis PCR was performed to mutate five individual amino acid residues in the ligand binding site of TAR. The primers that are utilized are the following:

TARR6973Fw:
gcgcaGGAAAGGTCTCACTGAGTnnNTCAGCGGTAnnNATGATGATGGATTCCTCCAATCAACAAAG
TARR6973Rv:
gcgcaGAGTAGGTCTCATCAGGTTAATGCGCGTTTGCAG
TARY149F150T154Fw:
gcgcaGGAAAGGTCTCAGGAGCTnnNnnNGCTCAGCCAnnNCAGGGAATGCAAAATGCAATGGGCGAAG
TARY149F150T154Rv:
gcgcaGAGTAGGTCTCACTCCAGTATTGCCATAATCTAGGTAATC

We firstly did a test PCR with both primer pairs to verify the mutagenesis PCR protocol and conditions. As a template we use the vector pSB1C3_TAR_QC_18C isolated from E. coli DH10B since this promoter is the strongest among the promoter constructs we use. After the amplification we have multiple linear vectors that contain every possible mutation.

Observations and results:
According to the agarose gel the PCR worked well. A band of the expected size of 3769 bp was observed for both reactions.

V07_03

Experiment:

Observations and results:

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