Team:Goettingen/week10-3

From 2012.igem.org

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<li>Observations and results: <br> Unfortunately we used the wrong amount of ethanol, so that the subsequent transformation did not work successfully.
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<li>Observations and results: <br> Unfortunately we used the wrong amount of ethanol, so that the subsequent transformation did not work successfully!
</li>
</li>
</ul>
</ul>
<b>V07_05_02 1<sup>st</sup> round: transforamtion of electrocompetent cells wir the mutant plasmid mixture</b><br>
<b>V07_05_02 1<sup>st</sup> round: transforamtion of electrocompetent cells wir the mutant plasmid mixture</b><br>
<ul>
<ul>
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<li>Experiment: <br>Electrocompetent cells were prepared according to <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. We transformed 200 µL of cells with 10 µL of DNA. A detailed protocol for the electroporation can be found <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">here</a>. The transformed cells were transferred to 100 mL liquid LB medium and incubated over night at 37 °C. We prepared three dilution plates (10<sup>4</sup>, 10<sup>5</sup>, 10<sup>6</sup>) for counting colonies to verify whether the desired library diversity is reached.
+
<li>Experiment: <br>Electrocompetent cells were prepared according to <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. We transformed 200 µL of cells with 10 µL of DNA. A detailed protocol for the electroporation can be found <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">here</a>. 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 (10<sup>4</sup>, 10<sup>5</sup>, 10<sup>6</sup>) for counting colonies to verify whether the desired library diversity is reached. Plates were incubated over night at 37 °C aswell.
</li>
</li>
</ul>
</ul>
<ul>
<ul>
-
<li>Observations and results: <br> Unfortunately we used the wrong amount of ethanol, so that the subsequent transformation did not work successfully.
+
<li>Observations and results: <br> Continuous bug see V07_05_01!
</li>
</li>
</ul>
</ul>

Revision as of 12:07, 22 September 2012

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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.


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 at 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 a EB volume of 50 µL.
  • Observations and results:
    The corresponding gel showed a band at 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!


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