Team:Concordia/ResultsCloning

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  • Lactococcus Lactis
  • Scaffold
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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 BBa_K1830000

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.