Team:Goettingen/week10-3

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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


1st round of mutagenesis PCR (mutation of aa residues 149, 150 and 159)
  • Experiment:
    The saturated mutagenesis PCR was set up in a 50 µL batch according to the established protocol V07_02).
  • Observations and results:
    The corresponding gel showed bands at the expected size.


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