Team:CSL Pittsburgh/Parts
Parts
"piGEM"
Part BBa_K112624 was obtained from the iGEM registry plates and transformed into competent E. coli. The transformants collected from the plate were subjected to colony PCR using VF2 and VR to verify the insert in the plasmid backbone. This part contains a pelB secretion signal and phoA reporter gene that we wanted to use for our final plasmid. To obtain these sequences we had to digest the plasmid with BamHI and NdeI; however, when we did this no band was observed containing the sequences of interest. To test why this occurred we digested the plasmid with each enzyme individually and observed that the BamHI restriction site was either not present or not functional.
Characterization of BBa_K112624
Figure 5. Characterization of part Part BBa_K112624 from the iGEM repository. A.)piGEM plasmid was double digested with BamHI and NdeI in cutsmart buffer to obtain the 1400 bp fragment containing phoA and pelB. However no band was observed. B.) The piGEM vector was digested with BamHI and NdeI separately and run on a gel with uncut plasmid. This gel shows that the BamHI site was not present or not functional.
pCYP6AY1
This plasmid, BBa_K3276005, was designed with the CYP6AY1 gene (a cytochrome P450) that was codon optimized from the brown plant hopper genome. This gene is constitutively driven by the promoter and RBS from part BBa_S05355. An NdeI and BamHI site were inserted upstream of the CYP6AY1 gene in order to add the pelB and phoA sequences from the piGEM plasmid. This synthesized oligo was ligated to the linearized pSB1C3 backbone and transformed into E. coli. Colonies from this transformation were collected and analyzed for the inserted DNA using the VF2 and VR primers.
pBee
This plasmid, BBa_K3276003, was designed with the CYP6AY1 gene (a cytochrome P450) that was codon optimized from the brown plant hopper genome. This gene is constitutively driven by the promoter and RBS from part BBa_S05355. A pelB secretion signal was added upstream of the CYP6AY1 gene and a 6x his tag was added to the end. This will allow the protein to be secreted from the cell and extracted and quantified. This synthesized oligo was ligated to the linearized pSB1C3 backbone and transformed into E. coli. Colonies from this transformation were collected and analyzed for the inserted DNA using the VF2 and VR primers. This is the main plasmid used in our studies due to the problems associated with the piGEM plasmid. The transformed E. coli was able to grow better in the presence of imidacloprid and helped the honey bees to survive exposure of the pesticide better than the wild type E. coli.
