Team:BrockU/Attributions
Team Achievements
Transforming pCDF-Duet-1 into Electrocompetent Cells. We used electrocompetent DH5α E. coli cells to amplify and plasmid extract the pCDF-Duet-1 plasmid. Robert, Raymond, Jenna, Sam, Michael, and Marvel were involved in this procedure.
Primer Design. We used the ApE software as well as the NEB Tm Calculator to design primers for the amplification of the native Flippase gene; as well as the FLPA, FLPB, pMag, and nMag fragments. Robert, Raymond, Jenna, Sam, Michael, and Marvel were involved in this procedure.
Restriction Enzyme Digest of pCDF-Duet-1. pCDF-Duet-1 was cut using the two restriction enzymes, NcoI and NotI. This enabled for subsequent Gibson Assembly. Sam, Michael, Matteo, and Marvel were involved in this procedure.
Amplification of the Flippase Gene. Using the primers created previously, we used PCR to amplify the flippase gene. The flippase fragment will be inserted into pCDF-Duet-1. Sam, Michael, and Marvel were involved in this procedure.
Gibson Assembly of Cut pCDF-Duet-1 and Flippase. Gibson Assembly was used to insert the flippase gene into the first multiple cloning site of pCDF-Duet-1. Sam, Michael, and Marvel were involved in this procedure.
Transforming pCDF-Duet-1-FLPinto Electrocompetent Cells. We used electrocompetent DH5α E. coli cells to amplify and plasmid extract the newly synthesized pCDF-Duet-1-FLP plasmid. Sam, Michael, and Marvel were involved in this procedure.
Cotransfection of pCDF-Duet-1-FLP and PKD4 Plasmids in E. coli cells. We used electrocompetent BL21DE3 E. coli cells to co-transfect the plasmids pCDF-Duet-1-FLP and PKD4. The BL21DE3 strain of E. coli was used since it possesses an inducible T7 polymerase. The colonies selected for positive double transformation were then used in the IPTG-mediated flippase induction assay. Sam, Michael, and Marvel were involved in this procedure.
IPTG-mediated Flippase Induction Assay. IPTG was used to induce the expression of flippase to assess the activity of the flippase enzyme. Sam, Michael, and Marvel were involved in this procedure.
Amplification of FLPA, FLPB, pMag, and nMag via PCR. Using the primers created previously, we used PCR to amplify these four fragments will be used in the cloning of Opto-FLP. Sam, Michael, and Marvel were involved in this procedure.
Primer Design. We used the ApE software as well as the NEB Tm Calculator to design primers for the amplification of the native Flippase gene; as well as the FLPA, FLPB, pMag, and nMag fragments. Robert, Raymond, Jenna, Sam, Michael, and Marvel were involved in this procedure.
Restriction Enzyme Digest of pCDF-Duet-1. pCDF-Duet-1 was cut using the two restriction enzymes, NcoI and NotI. This enabled for subsequent Gibson Assembly. Sam, Michael, Matteo, and Marvel were involved in this procedure.
Amplification of the Flippase Gene. Using the primers created previously, we used PCR to amplify the flippase gene. The flippase fragment will be inserted into pCDF-Duet-1. Sam, Michael, and Marvel were involved in this procedure.
Gibson Assembly of Cut pCDF-Duet-1 and Flippase. Gibson Assembly was used to insert the flippase gene into the first multiple cloning site of pCDF-Duet-1. Sam, Michael, and Marvel were involved in this procedure.
Transforming pCDF-Duet-1-FLPinto Electrocompetent Cells. We used electrocompetent DH5α E. coli cells to amplify and plasmid extract the newly synthesized pCDF-Duet-1-FLP plasmid. Sam, Michael, and Marvel were involved in this procedure.
Cotransfection of pCDF-Duet-1-FLP and PKD4 Plasmids in E. coli cells. We used electrocompetent BL21DE3 E. coli cells to co-transfect the plasmids pCDF-Duet-1-FLP and PKD4. The BL21DE3 strain of E. coli was used since it possesses an inducible T7 polymerase. The colonies selected for positive double transformation were then used in the IPTG-mediated flippase induction assay. Sam, Michael, and Marvel were involved in this procedure.
IPTG-mediated Flippase Induction Assay. IPTG was used to induce the expression of flippase to assess the activity of the flippase enzyme. Sam, Michael, and Marvel were involved in this procedure.
Amplification of FLPA, FLPB, pMag, and nMag via PCR. Using the primers created previously, we used PCR to amplify these four fragments will be used in the cloning of Opto-FLP. Sam, Michael, and Marvel were involved in this procedure.
Special Thanks
The BrockU iGEM would like to express our sincere gratitude to...
Taylor Lister and Devin Ward for providing constant mentorship and encouragement throughout the entire project.
Dr. Aleksandar Necakov for providing supervision, guidance and project support.
Jacinta Dano of Brock University for her encouragement, laboratory supplies and support.
Dr. Feng Li of Brock University for collaborating with our iGEM team and providing lab support.
Gregory Foran of Brock University for his project support and laboratory assistance.
Andrew Valente and Devin Ward for creating the BrockU iGEM wiki page.
Dr. Aleksandar Necakov, Devin Ward, and Taylor Lidster for presentation coaching.
Olivia Shaw-Shymanski for her support and for connecting our team with Youth in Science. We had the opportunity to discuss our projects to many youthful minds.
Carolyn Murphy for providing us with the opportunity to mentor young girls interested in pursuing their education in S.T.E.M.
Sultan Mussakhan, previous iGEM Gold medalist, for offering advice and guidance towards our project.
The MRT (Molecular Research Technologies) Club for collaborating with the iGEM team in order to promote molecular biology and our team project.
The Brock University Department of Chemistry for their project support.
The Brock University of Applied Health Sciences for their project support.
The Brock University Department of Biological Sciences for their project and financial support.
The Brock University Faculty of Math and Science for their project and financial support.
The Brock University Students’ Administrative Council (BUSAC) for their project and financial support.
E.S. Fox Limited for their interest in our project and their financial support.
New England BioLabs (NEB) for supplying the necessary reagents and supplies for our project.
Addgene for providing our team with the required project vectors.
SnapGene used to produce project illustrations.
Taylor Lister and Devin Ward for providing constant mentorship and encouragement throughout the entire project.
Dr. Aleksandar Necakov for providing supervision, guidance and project support.
Jacinta Dano of Brock University for her encouragement, laboratory supplies and support.
Dr. Feng Li of Brock University for collaborating with our iGEM team and providing lab support.
Gregory Foran of Brock University for his project support and laboratory assistance.
Andrew Valente and Devin Ward for creating the BrockU iGEM wiki page.
Dr. Aleksandar Necakov, Devin Ward, and Taylor Lidster for presentation coaching.
Olivia Shaw-Shymanski for her support and for connecting our team with Youth in Science. We had the opportunity to discuss our projects to many youthful minds.
Carolyn Murphy for providing us with the opportunity to mentor young girls interested in pursuing their education in S.T.E.M.
Sultan Mussakhan, previous iGEM Gold medalist, for offering advice and guidance towards our project.
The MRT (Molecular Research Technologies) Club for collaborating with the iGEM team in order to promote molecular biology and our team project.
The Brock University Department of Chemistry for their project support.
The Brock University of Applied Health Sciences for their project support.
The Brock University Department of Biological Sciences for their project and financial support.
The Brock University Faculty of Math and Science for their project and financial support.
The Brock University Students’ Administrative Council (BUSAC) for their project and financial support.
E.S. Fox Limited for their interest in our project and their financial support.
New England BioLabs (NEB) for supplying the necessary reagents and supplies for our project.
Addgene for providing our team with the required project vectors.
SnapGene used to produce project illustrations.