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:
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 of the expected size.
V07_04
V07_04_01 1st round: PCR clean-up
Experiment: The clean-up was performed using peqGOLD Cycle-Pure Kit (PeqLab) following the user manual. We modified the protocol by using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab) because these can bind a higher amount of DNA (30 µg/column). Thus, we lose fewer DNA material in the cleaning steps. 50 µL of elution buffer was used.
Observations and results: The corresponding gel showed a band of the expected size.
V07_04_02 1st round: DpnI/BsaI digestion
Experiment: The digestion was performed with a total volume of 50 µL according to protocol.
V07_04_03 1st round: Digestion clean-up
Experiment: The clean-up was performed using peqGOLD Cycle-Pure Kit (PeqLab) modified by using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab) with an EB volume of 50 µL.
Observations and results: The corresponding gel showed a band of the expected size.
V07_04_04 1st round: Ligation
Experiment: The ligation was set up in a 100 µL batch according to protocol. Incubation over night at 16 °C.
V07_05
V07_05_01 1st round: Ethanol precipitation of mutated plasmids
Experiment: The ethanol precipitation was performed according to protocol.
Observations and results: Unfortunately we used the wrong amount of ethanol, so that the subsequent transformation did not work successfully!
V07_05_02 1st round: Transforamtion of electrocompetent cells wir the mutant plasmid mixture
Experiment: Electrocompetent cells were prepared according to protocol. We transformed 200 µL of cells with 10 µL of DNA. A detailed protocol for the electroporation can be found here. The transformed cells were transferred to 100 mL liquid LB medium and incubated over night at 37 °C, 180 rpm. We prepared three dilution plates LB+CM (104, 105, 106) for counting colonies to verify whether the desired library diversity is reached. Plates were incubated over night at 37 °C aswell.
Observations and results: Continuous bug see V07_05_01!
V07_06
V07_06_01 1st round: Analysis of transformation V07_05
Experiment: Plates and liquid culture were checked for successful transformation.
Observations and results: No bacterial growth on neither plates nor in liquid culture. Continuous bug see V07_05_01!
V07_06_02 1st round: Restart of saturated mutagenesis PCR
Experiment:
Observations and results: The corresponding gel showed bands of the expected size.
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