Team:USMA-West Point/Basic Part

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USMA-West_Point

<!DOCTYPE html> About - iGem 01

usMA-WEST POINT


Basic Parts




Plasmid design and development is a key component of the eNose project. Since odorant receptor protein function was tested in a mammalian cell line, the plasmids used to drive expression of these proteins were specifically designed for mammalian cells. For example, the odorant receptor proteins OR10G4 and OR2W1 were expressed using the pCMV6 and pCI plasmids, respectively, and both contain a cytomegalovirus (CMV) promoter region for high gene expression levels in mammalian cells.




For construction of iGEM deliverables containing odorant receptor proteins in the pSB1C3 plasmid, the coding sequences for OR10G4 and OR2W1 were amplified by PCR using the respective pCMV6 and pCI mammalian expression plasmids as templates. The PCR reactions were expected to generate bands of approximately 0.95kb for both OR10G4 and OR2W1 coding sequences, and the image of the gel for the PCR products shows bands at this expected size. These PCR products were amplified using primers that incorporated EcoRI and PstI restriction endonuclease sites at the 5’ and 3’ ends of the PCR products. Digesting both the PCR products and the pre-linearized pSB1C3 plasmid supplied by iGEM with EcoRI and PstI restriction endonucleases, the PCR products were ligated into the pSB1C3 plasmid. The ligation reaction was used to transform chemically competent DH5alpha cells. To screen for colonies containing the appropriately inserted odorant receptor gene and pSB1C3 plasmid, colonies were used to inoculate small cultures. The plasmids were harvested from the cultures by mini-prep plasmid isolation kits, and the isolated plasmids were digested with two sets of restriction endonuclease reactions.





For the OR2W1 in pSB1C3 plasmid, one reaction used the BglII and PstI enzymes. The expected products for this reaction were two bands at 2.3kb and 0.8kb. Because the BglII site is unique to OR2W1 coding sequence, then only a single band at 2.2kb would be expected if the colony contained only the empty pSB1C3 plasmid. The second reaction used the MscI enzyme. The expected products for this reaction were also two bands at 1.9kb and 1.2kb. Because both the pSB1C3 plasmid and OR10G4 coding sequence each contain a single MscI site, then only a single band at 2.2kb would be expected if the colony contained only the empty pSB1C3 plasmid. As shown by the image of the gel with the restriction endonuclease reaction products, the band patterns reveal a colony containing the pSB1C3 plasmid with the OR2W1 coding sequence.




For the OR10G4 in pSB1C3 plasmid, one reaction used the EcoRI and BamHI enzymes. The expected products for this reaction were two bands at 2.3kb and 0.7kb. Because the BamHI site is unique to OR10G4 coding sequence, then only a single band at 2.2kb would be expected if the colony contained only the empty pSB1C3 plasmid. The second reaction used the BlpI and PstI enzymes. The expected products for this reaction were also two bands at 2.3kb and 0.7kb. Because the BlpI site is unique to OR10G4 coding sequence, then only a single band at 2.2kb would be expected if the colony contained only the empty pSB1C3 plasmid. As shown by the image of the gel with the restriction endonuclease reaction products, the band patterns reveal a colony contains the pSB1C3 plasmid with the OR10G4 coding sequence.




DEPARTMENT OF CHEMISTRY AND LIFE SCIENCE



BARTLETT HALL, ROOM 400, WEST POINT, NY 10996, USA


TEL: 845.938.3915