Difference between revisions of "Team:UGent Belgium/LabNotebook"

• 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. The inaZ gene was sourced from BBa_K584027 using primers oM7628 and oM7629* (length 3.6 kbp, anneal at 58°C), transformed into E. coli DH5a cells by heatshock, and plated on chloramphenicol. The inaX gene was sourced from X. campestris colonies using primers oM7626 and oM7627 (length 4.7 kbp, anneal at 70°C).

  
  
  
  
  
  

• We transformed the strong expression vector (cf. 29/08) by electroshock into E. coli Top10, and we plated it on chloramphenicol.

• As a backup strategy we also used CPEC with the backbone from the K584027 plasmid to generate a strong expression vector. For this, we used Primestar HS PCR with primers oM7643 and oM7645.

• Second attempt (cf. 29/08) to isolate the inaZ gene from P. syringae and the inaX gene from X. campestris. InaX failed, but inaZ was successful. Annealing temperature was 66.20°C and 61.93°C for respectively both primers for the inaZ gene, 60.96°C and 70.34°C for respectively both primers of the inaX gene. Both Primestar HS and Primestar GXL PCR were used for this. On the gel, lane 4 and 8 are inaX attempts.

  
  
  
  
  
  

• 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 (see gel, cf. 30/08).

  
  
  
  
  
  

• Heatshock transformation of the strong expression vector made with CPEC (cf. 31/08) 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). As primers, we used BioBrick verification primers.

Electroshock transformation of the strong expression vector made with CPEC (cf. 31/08) into E. coli Top10 cells.

• 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 used BioBrick verification primers for this.

  
  
  
  
  
  

• 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).

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

We transformed our Golden Gate reactions (cf. 06/08) into E. coli Top10 cells.

• 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 internal BsaI sites.

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 repeated the colony PCR under different conditions, but bands were still not of the correct lengths. 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

• 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).

• All our plates with the weak expression vector constructs show colonies! The control reaction has colonies as well, but much fewer, which was to be expected. Fluorescence is seen for the pXW-INP_NC-mGFPuv construct. All Lpp-ompA plates have a mixed phenotype, with 10-20 large colonies (same size as control reaction), and >60 small colonies (see culture picture). Colony PCR was performed using OneTaq at 50°C annealing temperature to proof that the small colonies are the positives ones, the larger ones the negative ones. This theory was confirmed for all colonies (see gel picture).

  
  
  
  

  
  
  

• We performed Primestar HS PCR to create following constructs:

  • pXW-Lpp-ompA-mGFPuv-(m)Strep using Lpp-ompA-mGFPuv fragment. As a template, we used the GG mix for pXW-Lpp-ompA-mGFPuv, and oMEMO7667_Lpp_Fw and oMEMO7663_GFP_Rv-ATCT as primers (expected length:1167).

  • pXW-mGFPuv-m(Strep) using mGFP. As a template, we used a 100x diluted gBlock, and oMEMO7662_GFP_Fw-GATG and oMEMO7663_GFP_Rv-ATCT as primers (expected length: 752 bp).

  • pXS-mGFPuv using mGFP. As a template, we used a 100x diluted gBlock, and oMEMO7662_GFP_Fw-GATG and oMEMO7664_GFP_Rv-TACT as primers (expected length: 752 bp).

  We also repeated previous PCR (cf. 08/09) of the largest piece of inaZ to make it Golden Gate safe. As a template, we used the inaZ plasmid, and oM7658 and oM7629 as primers (expected length: 3039 bp).

• 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

• 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.

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

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

• 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.

• 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.

• 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.

• 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.

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

Colony PCR of all CPEC assemblies (cf. 26/09).

• Second attempt for CPEC assemblies (cf. 26/09), with increased backbone DNA and an altered backbone/insert ratio.
• Primestar HS PCR to create GATG-mGFPuv-ATCT, which is necessary for new attempts for the pXW-mGFPuv-Strep and pXW-mGFPuv-mSA2 constructs, due to mutations in previous versions.
• Golden Gate assemblies to create:
  • pXW-inaZ-RC+Strep
  • pXW-inaZ-RC+mSA2
  • pXW-inaZ-RC+GFP
  • pXW-mGFPuv-Strep
  • pXS-mGFPuv-Strep
  • pXS-mGFPuv-mSA2
• We also started cultures for the cell binding assay to test the attachment to our filament, and for protein extraction. For the first, we used constructs pXW-INP_NC-Strep + pSC101-RFP, pXW-INP_NC-mSA2 + pSC101-RFP, and pSC101-RFP. For the latter, we used constructs pXW-mGFPuv-mSA2 and pXW-mGFPuv.

• Sequenced two new colonies of construct pXW-mGFPuv-Strep (due to mutation in GFP).
• Created pSB1C3-mGFPuv2 for submission to the parts registry. For this we amplified the backbone from pSB1C3 linear template using primers oM7722 and oM7723; and mGFPuv2 from construct or using primers oM7662 and oM7664. Golden Gate assembly was done using BsaI.

We did colony PCR on the CPEC assemblies with increased backbone DNA and altered backbone/insert ratio (cf. 28/09), and previous Golden Gate assemblies (cf. 28/09). We found positive colonies for following constructs:

• pLC-XW
• pLC-XW-INP_NC-Strep
• pLC-XW-INP_NC-mSA2
• pXW-inaZ-RC+mSA2
• pXW-mGFPuv-Strep
• pXS-mGFPuv-Strep
• pXS-mGFPuv-mSA2

New colony PCR of colonies of constructs pXW-inaZ-RC+Strep and pXW-inaZ-RC+GFP.

We found a positive colony for construct pXW-inaZ-RC+GFP (cf. 03/10).

Golden Gate assembly of pSB1C3-mGFPuv2, with a shorter protocol (cf. 29/09). It was then transformed into E. coli Top10 cells.

• Colony PCR on pSB1C3-mGFPuv2 colonies (cf. 05/10). For primers, we used biobrick verification primers. Expected length is 1030 bp.
• Combined pXW-INP_GGsafe with pLC-XW-INP_NC-mSA2 by electroporation (plate on kan+chloramphenicol).