Team:UGent Belgium/LabNotebook

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Lab Notebook

August

  • August 29:

    • We made a strong expression vector (pXS) by restriction of the linearized plasmid backbone pSB1C3 and insertion of gblock made up of promoter-RBS-cloning site-terminator-terminator (cut with EcoRI and PstI).
    • Our first attempts to isolate the inaZ gene from P. syringae and the inaX gene from X. campestris failed.
  • August 30:

    • We transformed the strong expression vector (cf. 29/08) by electroshock into E. coli TOP10.
    • As a backup strategy we also used CPEC with the backbone from the K584027 plasmid to generate a strong expression vector.
  • August 31:

    • Second attempt to isolate the inaZ gene from P. syringae and the inaX gene from X. campestris. InaX failed, but inaZ was successful.
    • Transformation of the strong expression vector by restriction and ligation was not very successful, we only had a few colonies. The backbone amplification using CPEC was successful (cf. 30/08).

September

  • September 01:

    • Heatshock transformation of the strong expression vector made with CPEC yielded no colonies.
    • Colony PCR on the colonies with the strong expression vector made with restriction and ligation didn’t show any positive ones (cf. 31/08).
  • September 02:

    Electroshock transformation of the strong expression vector made with CPEC into E. coli TOP10 cells.

  • September 04:

    • Via colony PCR we saw that all colonies with the strong expression vector made with CPEC and electroshock transformation were all positive (cf. 02/09).
    • We transformed E. coli TOP10 cells with our weak expression vector (pXW), which came in today. Q5 CPEC was used for this with the linearized plasmid backbone pSB1C3 and as insert a gBlock consisting of promoter-RBS-cloning site-terminator-terminator. The promoter is weaker than the promoter of the strong expression vector (cf. 29/08).