Difference between revisions of "Team:Concordia/ResultsCloning"
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<img src="https://static.igem.org/mediawiki/2015/5/54/Concordia-pet15bb.png" class= "img-thumbnail" alt="pet15BB line" align="left" width=50%" style="margin-right: 20px"> | <img src="https://static.igem.org/mediawiki/2015/5/54/Concordia-pet15bb.png" class= "img-thumbnail" alt="pet15BB line" align="left" width=50%" style="margin-right: 20px"> | ||
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Expression vectors are a basic necessity for the production of large amounts of proteins in vivo. An expression vector contains the appropriate promoter, RBS, and terminator for use in the host cell. <br><br> | Expression vectors are a basic necessity for the production of large amounts of proteins in vivo. An expression vector contains the appropriate promoter, RBS, and terminator for use in the host cell. <br><br> | ||
The pET expression system is a universal system used for protein expression. This expression system, adopted from the pBR322 plasmid, uses the T7 RNA polymerase gene which is unique to the T7 lambda phage, and an inducible LacI gene. Thus, the gene of interest is cloned downstream of a unique promoter and its expression is under the control of an inducer such as IPTG.<br><br> | The pET expression system is a universal system used for protein expression. This expression system, adopted from the pBR322 plasmid, uses the T7 RNA polymerase gene which is unique to the T7 lambda phage, and an inducible LacI gene. Thus, the gene of interest is cloned downstream of a unique promoter and its expression is under the control of an inducer such as IPTG.<br><br> | ||
Revision as of 04:21, 21 November 2015
Results of Cloning
Gel Electrophoresis of Team Parts
We successfully cloned the 8 components (registry parts BBa_K1830001 to BBa_K1830008) making up the scaffold, as seen in gel lanes 1 to 16, into a single construct and the 2 dockerin parts Dock1 and XDock2 (BBa_K1830009 and BBa_K1830010), as seen in lanes 17 to 20, into their respective pSB1C3 backbones. Gel lanes 21 to 26 showcase 3 enzymes, SacC, ADH2, ALD2, that were cloned into backbones. In total, 13 original parts were added by our team to the iGEM Parts registry.
| Gel Lanes | Part | Registry # |
|---|---|---|
| 1,2 | PnisA | BBa_K1830001 |
| 3,4 | SPusp45 | BBa_K1830002 |
| 5,6 | CBD | BBa_K1830003 |
| 7,8 | Coh1c3 | BBa_K1830004 |
| 9,10 | Coh2S1 | BBa_K1830005 |
| 11,12 | CWAm6 | BBa_K1830006 |
| 13,14 | T1T2 terminator | BBa_K1830007 |
| 15,16 | Lactis shuttle | BBa_K1830008 |
| 17,18 | Dock1 | BBa_K1830009 |
| 19,20 | XDock2 | BBa_K1830010 |
| 21,22 | SacC | BBa_K1830015 |
| 23,24 | ADH2 (alcohol dehydrogenase) | BBa_K1830013 |
| 25,26 | ALD2 (aldehyde dehydrogenase) | BBa_K1830014 |
pET15BB Expression vector
Expression vectors are a basic necessity for the production of large amounts of proteins in vivo. An expression vector contains the appropriate promoter, RBS, and terminator for use in the host cell.
The pET expression system is a universal system used for protein expression. This expression system, adopted from the pBR322 plasmid, uses the T7 RNA polymerase gene which is unique to the T7 lambda phage, and an inducible LacI gene. Thus, the gene of interest is cloned downstream of a unique promoter and its expression is under the control of an inducer such as IPTG.
Due to the specific conditions for iGEM part submission and a dearth of vectors with RFC compatible cut sites, teams must currently perform additional steps in order to have their part expressed with the pET plasmid. The pET15BB expression vector hopes to minimize these steps by providing a backbone which retains the key characteristics of the pET expression system, but with the addition of an MCS with RFC 25 compatible cut sites.