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).
  • September 06:

    Colonies from our strong expression vector made with CPEC (cf. 04/09) are cultured and miniprepped. These are used for following constructs:

    • pXS-INP_WT using the inaZ PCR fragment
    • pXS-INP_NC-mGFPuv
    • pXS-INP_NC-Strep (Strep: regular streptavidin)
    • pXS-INP_NC-mSA2 (mSA2: monomeric streptavidin)
    • pXS-Lpp-ompA-mGFPuv
    • pXS-Lpp-ompA-Strep
    • pXS-Lpp-ompA-mSA2
    All constructs are made by using a Golden Gate reaction mix.
  • September 07:

    We transformed our Golden Gate reactions (cf. 06/08) into E. coli TOP10 cells.

  • September 08:

    • We did a colony PCR of our weak expression vector (cf. 04/09), and of our constructs made with the strong expression vector (cf. 06/09), both using BioBrick verification primers.
    • We tried to make inaZ Golden Gate safe by removing the BsaI sites. To achieve this, the gene is cut into 3 fragments. The combined fragments however did not show the correct length.
  • September 09:

    The colony PCR of E. coli cells with the constructs with the strong expression vector showed no positive colonies (cf. 06/09). The colony PCR on our cells transformed with the weak expression vector (cf. 04/09), however, did. We therefore tried cloning all our parts into the weak expression vector by using a Golden Gate reaction mix. These Golden Gate reactions were then transformed into E. coli TOP10 cells. Incubation was done at 30°C instead of 37°C to repress the plasmid copy number

  • September 10:

    • We got our sequencing results: the strong expression vector is good!
    • Colonies with the weak expression vector constructs are still too small to pick up with colony PCR (cf. 09/09)
  • September 11:

    • All our plates with the weak expression vector constructs show colonies! Fluorescence is seen for the pXW-INP_NC-mGFPuv construct. All Lpp-ompA plates have a mixed phenotype, and colony PCR was performed to proof that the small colonies are the positives ones, the larger ones the negative ones.
    • We performed PCR to create following constructs:
      • pXW-Lpp-ompA-mGFPuv-(m)Strep using Lpp-ompA-mGFPuv fragment
      • pXW-mGFPuv-m(Strep) using mGFP
      • pXS-mGFPuv using mGFP
      We also repeated previous PCR (cf. 08/09) of the largest piece of inaZ to make it Golden Gate safe.
  • September 12:

    • Miniprep of a number of colony PCR positive colonies (cf. 09/09).
    • Golden Gate assemblies of:
      • pXW-inaZ_GGsafe
      • pXW-mGFPuv-Strep
      • pXW-mGFPuv-mSA2
      • pXW-mGFPuv
      • pXS-mGFPuv
  • September 13:

    • pXW-Lpp-ompA-mGFPuv miniprep tubes that did not grow yesterday did show growth today, probably through mutations.
    • Transformed golden gate assemblies (cf. 12/09) into E. coli TOP10 cells.
  • September 14:

    Colony PCR of the Golden Gate assemblies (cf. 12/09): fail for pXW-INP_GGsafe, okay for other assemblies

  • September 15:

    Colony PCR screening of 8 more colonies with pXW-inaZ_GGsafe showed no result (cf. 13/09)

  • September 16:

    • Minipreps of the following plasmids and sequencing with verification primers:
      • pXW-mGFPuv-Strep
      • pXW-mGFPuv-mSA2
      • pXW-mGFPuv
      • pXS-mGFPuv
    • We did a PCR to go from high copy to low copy (LC) backbone, with as backbone pSB3K3 and 4 inserts: INP_NC-Strep, INP_NC-mSA2, Lpp-ompA-Strep and Lpp-ompA-mSA2. The backbone and Lpp-ompA-mSA2 both failed.
  • September 19:

    • We retried the PCR to go from high to low copy backbone, with new primers for the pSB3K3 backbone, but this again failed. The Lpp-ompA-mSA2 insert was also redone with now the Golden Gate mix as template, which was successful.
    • Golden gate assembly of pXW-inaZ_GGsafe.
  • September 21:

    • We did a colony PCR for the Golden Gate assembly of pXW-inaZ_GGsafe (cf. 19/09).
    • We opted for a new backbone for the low copy vector (cf. 19/09): pSC101-Kan vector.
  • September 22:

    • We digested some colony PCR samples (cf. 21/09), and controlled them with BsaI. If pXW-inaZ_GGsafe is indeed Golden Gate safe, it should not be cut. This was okay for two colonies! These colonies are plasmid prepped for sequencing.
    • We again retried the PCR to go from high to low copy backbone.For this, we used pSC101 as backbone. A new Lpp-ompA-mSA2 insert was also done, with again the Golden Gate mix as template. A weak expression vector with an empty expression site was also added.
  • September 26: CPEC assemblies to create:
    • pLC-XW
    • pLC-XW-INP_NC-Strep
    • pLC-XW-INP_NC-mSA2
    • pLC-XW-Lpp-ompA-Strep
    • pLC-XW-Lpp-ompA-mSA2
    • pSB1C3-mSA2