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. Marvel, Taylor, and Devin 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. Marvel, Taylor, and Devin 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. Marvel, Taylor, and Devinwere 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. Marvel, Taylor, and Devin were involved in this procedure.
Completing the Opto-FLP Construct via Gibson Assembly. The amplified fragments: FLPA, FLPB, pMag and nMag were all inserted into pCDF-Duet-1 using Gibson Assembly to create pCDF-Duet-1-Opto-FLP. Marvel, Taylor, and Devin were involved in this procedure.
Designing FRT-mScarlett-STOP-FRT Fragment. This fragment was used for the colour switch assay. It was designed by the team and synthesized by IDT. Heather, Josh, and Shraddha were involved in this procedure.
Restriction Enzyme Digest of pRSET-mSA-eGFP Plasmid. The pRSET-mSA-eGFP plasmid was digested with NcoI and XhoI to linearize it for the T4 ligation described below. Brad and Stephanie were involved in this procedure.
Restriction Enzyme Digest of FRT-mScarlet-STOP-FRT Fragment. The IDT synthesized FRT-mScarlet-STOP-FRT fragment was digested with NcoI and XhoI to prepare it for the T4 ligation described below. Brad and Stephanie were involved in this procedure.
Constructing the pRSET-FRT-mScarlet-STOP-FRT-eGFP Plasmid. Using T4 ligation, FRT-mScarlet-STOP-FRT and the pRSET-eGFP plasmid were ligated together to create the pRSET-FRT-mScarlet-STOP-FRT-eGFP (colour-switch) plasmid. Heather, Josh, and Shraddha were involved in this procedure.
Testing the Reporter pRSET-FRT-mScarlet-STOP-FRT-eGFP.. BL21 DE3 E. coli cells were transformed with the plasmid and grown up in liquid culture. IPTG was used to induce transcription. Samples of different time points after induction were taken and the cells were imaged using the Zeiss Observer Z1 inverted fluorescence microscope to visualize mScarlet and eGFP protein expression levels. Josh, Brad, Shraddha, Matthew, Stephanie, and Brandon were involved in this procedure.
Testing the Opto-FLP Construct Using the Colour Switch Assay. BL21 DE3 E. coli cells were co-transformed with pCDF-Duet-1-Opto-FLP and pRSET-FRT-mScarlet-STOP-FRT-eGFP. IPTG was used to induce transcription. Cells from different time points post IPTG addition were imaged using the fluorescence microscope to visualize mScarlet and eGFP protein expression levels and determine the functionality of Opto-FLP. Heather, Josh, Josh, Brad, Shraddha, Matthew, Stephanie, Meagan, and Brandon 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. Marvel, Taylor, and Devin 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. Marvel, Taylor, and Devinwere 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. Marvel, Taylor, and Devin were involved in this procedure.
Completing the Opto-FLP Construct via Gibson Assembly. The amplified fragments: FLPA, FLPB, pMag and nMag were all inserted into pCDF-Duet-1 using Gibson Assembly to create pCDF-Duet-1-Opto-FLP. Marvel, Taylor, and Devin were involved in this procedure.
Designing FRT-mScarlett-STOP-FRT Fragment. This fragment was used for the colour switch assay. It was designed by the team and synthesized by IDT. Heather, Josh, and Shraddha were involved in this procedure.
Restriction Enzyme Digest of pRSET-mSA-eGFP Plasmid. The pRSET-mSA-eGFP plasmid was digested with NcoI and XhoI to linearize it for the T4 ligation described below. Brad and Stephanie were involved in this procedure.
Restriction Enzyme Digest of FRT-mScarlet-STOP-FRT Fragment. The IDT synthesized FRT-mScarlet-STOP-FRT fragment was digested with NcoI and XhoI to prepare it for the T4 ligation described below. Brad and Stephanie were involved in this procedure.
Constructing the pRSET-FRT-mScarlet-STOP-FRT-eGFP Plasmid. Using T4 ligation, FRT-mScarlet-STOP-FRT and the pRSET-eGFP plasmid were ligated together to create the pRSET-FRT-mScarlet-STOP-FRT-eGFP (colour-switch) plasmid. Heather, Josh, and Shraddha were involved in this procedure.
Testing the Reporter pRSET-FRT-mScarlet-STOP-FRT-eGFP.. BL21 DE3 E. coli cells were transformed with the plasmid and grown up in liquid culture. IPTG was used to induce transcription. Samples of different time points after induction were taken and the cells were imaged using the Zeiss Observer Z1 inverted fluorescence microscope to visualize mScarlet and eGFP protein expression levels. Josh, Brad, Shraddha, Matthew, Stephanie, and Brandon were involved in this procedure.
Testing the Opto-FLP Construct Using the Colour Switch Assay. BL21 DE3 E. coli cells were co-transformed with pCDF-Duet-1-Opto-FLP and pRSET-FRT-mScarlet-STOP-FRT-eGFP. IPTG was used to induce transcription. Cells from different time points post IPTG addition were imaged using the fluorescence microscope to visualize mScarlet and eGFP protein expression levels and determine the functionality of Opto-FLP. Heather, Josh, Josh, Brad, Shraddha, Matthew, Stephanie, Meagan, and Brandon were involved in this procedure.
Special Thanks
The BrockU iGEM team would like to express our sincere gratitude to...
Greg Foran, Taylor Lister, Marvel Magely, and Devin Ward for providing constant mentorship and encouragement throughout the entire project.
Matteo Andrade, Michael Palmieri, and Samantha Toole for their previous work on this 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.
Andrew Valente and Devin Ward for creating the BrockU iGEM wiki page.
Matteo Andrade for collaborating with the iGEM team to provide guidance of bureaucracy and obtaining funding.
Dr. Aleksandar Necakov, Taylor Lister, Marvel Magely, and Devin Ward for presentation coaching.
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 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.
Palfinger Inc. for their project and financial support.
Java Jacks Restaurant and Gallery for their project and financial support.
Niagara Automotive for their project and financial support.
Integrated DNA Technologies (IDT) for donating various unique DNA oligonucleotides.
New England BioLabs (NEB) for donating the necessary reagents and supplies for our project.
Addgene for providing our team with the required project vectors.
SnapGene used to produce project illustrations.
The BrockU iGEM team would like to express a special thank you to our donors:
Greg Foran, Taylor Lister, Marvel Magely, and Devin Ward for providing constant mentorship and encouragement throughout the entire project.
Matteo Andrade, Michael Palmieri, and Samantha Toole for their previous work on this 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.
Andrew Valente and Devin Ward for creating the BrockU iGEM wiki page.
Matteo Andrade for collaborating with the iGEM team to provide guidance of bureaucracy and obtaining funding.
Dr. Aleksandar Necakov, Taylor Lister, Marvel Magely, and Devin Ward for presentation coaching.
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 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.
Palfinger Inc. for their project and financial support.
Java Jacks Restaurant and Gallery for their project and financial support.
Niagara Automotive for their project and financial support.
Integrated DNA Technologies (IDT) for donating various unique DNA oligonucleotides.
New England BioLabs (NEB) for donating the necessary reagents and supplies for our project.
Addgene for providing our team with the required project vectors.
SnapGene used to produce project illustrations.
The BrockU iGEM team would like to express a special thank you to our donors: