Difference between revisions of "Team:Fudan-TSI/Part Collection"

Fudan-TSI provides you with a toolbox for in vivo site-specific continuous mutagenesis. The pivotal elements in the collection are Cre recombinase and Moloney Murine Leukemia Virus reverse transcriptase (MMLV-RT). Our collection contains Cre attached with different degradation tags, and RT under different inducible promoters to achieve diverse steady-state expression level. All parts are well characterized, for researchers to choose their desired ones. To initiate reverse transcription and recombination, additional regulatory sequences are required. We provide incompatible loxP sites for Cre and primer binding site along with other reverse transcription completion sequences for RT. We determined our most favored construct based on both the experimental and modelling result. Our collection provides researchers with a set of elements necessary for mutagenesis, and they can assemble the system based on their own needs.

In order to produce the reverse transcriptase effectively inside E.coli cells, we have constructed a series of promoters expressing gag-pol polyprotein with or without operons controlling the transcription. Cloned into a proper protein expression vector, we believe that reverse transcriptase can be expressed in E.coli.

Part Number	Part Name	Link
BBa_K3257106	gag-Q-pol with T5 Promoter	http://parts.igem.org/Part:BBa_K3257106
BBa_K3257107	gag-Q-pol with T7 Promoter	http://parts.igem.org/Part:BBa_K3257107
BBa_K3257108	gag-Q-pol with J23119 Promoter	http://parts.igem.org/Part:BBa_K3257108
BBa_K3257109	gag-Q-pol with T8 Promoter	http://parts.igem.org/Part:BBa_K3257109
BBa_K3257110	gag-Q-pol with T9 Promoter	http://parts.igem.org/Part:BBa_K3257110

This collection includes the components necessary for the reverse transcription initiation, in which tRNAPro and PBS acts as a primer and its binding site, and others are involved in the first and second strand transfer process.

Part Number	Part Name	Link
BBa_K3257060	PolyPurine Tract(PPT)	http://parts.igem.org/Part:BBa_K3257060
BBa_K3257061	R Region	http://parts.igem.org/Part:BBa_K3257061
BBa_K3257062	U5	http://parts.igem.org/Part:BBa_K3257062
BBa_K3257063	Primer Binding Site(PBS)	http://parts.igem.org/Part:BBa_K3257063
BBa_K3257076	tRNA Primer	http://parts.igem.org/Part:BBa_K3257076

This collection includes 5 different degradation tags with different degradation rate and steady-state concentration when fused after a CDS of a protein. We have measured the induction curve and growth curve of BL21 (DE3) containing EGFP fused with degradation tags (Figure 1).

Part Number	Part Name	Link
BBa_K3257069	SsrA(LAA)	http://parts.igem.org/Part:BBa_K3257069
BBa_K3257070	SsrA(LAV)	http://parts.igem.org/Part:BBa_K3257070
BBa_K3257071	SsrA(LVA)	http://parts.igem.org/Part:BBa_K3257071
BBa_K3257072	SsrA(LVV)	http://parts.igem.org/Part:BBa_K3257072
BBa_K3257073	SsrA(WVLAA)	http://parts.igem.org/Part:BBa_K3257073

Combined with Degradation Tags Collection, the leakage of Cre recombinase is solved and difference in expression level between Cre and RT is achieved in an easier way. The steady state concentration of Cre recombinase lowers to a functional level.

Part Number	Part Name	Link
BBa_K3257101	Cre with SsrA(WVLAA)	http://parts.igem.org/Part:BBa_K3257101
BBa_K3257102	Cre with SsrA(LAA)	http://parts.igem.org/Part:BBa_K3257102
BBa_K3257103	Cre with SsrA(LAV)	http://parts.igem.org/Part:BBa_K3257103
BBa_K3257104	Cre with SsrA(LVA)	http://parts.igem.org/Part:BBa_K3257104
BBa_K3257105	Cre with SsrA(LVV)	http://parts.igem.org/Part:BBa_K3257105

This collection includes 4 different types of lox sites. The lox511, lox2272, lox5171 are all orthogonal to loxP as we can see from their inability to be knocked out by Cre if there are loxP on one side and other lox sites on the other side.

After double transformation, colony PCR was done and agarose gel shows that there remains the original DNA fragment.

Part Number	Part Name	Link
BBa_K3257064	loxP	http://parts.igem.org/Part:BBa_K3257064
BBa_K3257065	loxP511	http://parts.igem.org/Part:BBa_K3257065
BBa_K3257074	lox2272	http://parts.igem.org/Part:BBa_K3257074/td>
BBa_K3257075	lox5171	http://parts.igem.org/Part:BBa_K3257075