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	Notes
BBa_K3257106	gag-Q-pol with T5 Promoter	IPTG Inducible
BBa_K3257107	gag-Q-pol with T7 Promoter	IPTG Inducible
BBa_K3257108	gag-Q-pol with J23119 Promoter	IPTG Inducible
BBa_K3257110	gag-Q-pol with LacUV5 Promoter	IPTG Inducible

This collection includes wild-type Chloramphinicol resistence gene and its 7 nonsense mutants, which are used as targets of our in vivo sequence-specific toolbox for continuous mutagenesis system. By multiple rounds of mutagenesis process, nonsense mutated Chl^R gene will undergo recovery mutation and regain its ability of Chloramphinicol resistence.

Part Number	Part Name	Notes
BBa_K3257034	ChlR	Wild-type ChlR gene.
BBa_K3257035	ChlR_T33X	T33X mutant of ChlR gene.
BBa_K3257036	ChlR_Y92X	Y92X mutant of ChlR gene.
BBa_K3257037	ChlR_E97X	E97X mutant of ChlR gene.
BBa_K3257038	ChlR_S121X	S121X mutant of ChlR gene.
BBa_K3257039	ChlR_L158X	L158X mutant of ChlR gene.
BBa_K3257040	ChlR_K182X	K182X mutant of ChlR gene.
BBa_K3257041	ChlR_Q190X	Q190X mutant of ChlR gene.

This collection includes the components necessary for the reverse transcription initiation, in which tRNAP^ro 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	Notes
BBa_K3257060	PolyPurine Tract(PPT)	Involved in the first strand transfer in reverse transcription process
BBa_K3257061	R Region	Involved in the first strand transfer in reverse transcription process
BBa_K3257062	U5 Region	Important for primer binding and virus encapsidation
BBa_K3257063	Primer Binding Site(PBS)	Providing bingding site for reverse transcriptase
BBa_K3257076	tRNA Primer	Initiating the reverse transcription process

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	Notes
BBa_K3257069	SsrA(LAA)	LAA stands for the last 3 amino acid residues of this degradation tag and this is a widely-used wild-type tag.
BBa_K3257070	SsrA(LAV)	LAV stands for the last 3 amino acid residues of this degradation tag and it is mutated from SsrA(LAA).
BBa_K3257071	SsrA(LVA)	LVA stands for the last 3 amino acid residues of this degradation tag and it is mutated from SsrA(LAA).
BBa_K3257072	SsrA(LVV)	LVA stands for the last 3 amino acid residues of this degradation tag and it is mutated from SsrA(LAA).
BBa_K3257073	SsrA(WVLAA)	WVLAA stands for the last 5 amino acid residues of this degradation tag and it is mutated from SsrA(LAA).

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	Notes
BBa_K3257101	Cre with SsrA(WVLAA)	Cre recombinase tightly attached with SSr(WVLAA)
BBa_K3257102	Cre with SsrA(LAA)	Cre recombinase tightly attached with SSr(LAA)
BBa_K3257103	Cre with SsrA(LAV)	Cre recombinase tightly attached with SSr(LAV)
BBa_K3257104	Cre with SsrA(LVA)	Cre recombinase tightly attached with SSr(LVA)
BBa_K3257105	Cre with SsrA(LVV)	Cre recombinase tightly attached with SSr(LVV)

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	Notes
BBa_K3257064	loxP	Wild-type lox site that can be recognized by Cre recombinase.
BBa_K3257074	lox2272	Orthogonal lox site mutated from loxP.
BBa_K3257075	lox5171	Orthogonal lox site mutated from loxP.