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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).
 
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).
 
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<div class="col">The Cre Recombinase with Degradation Tags Collection</div>
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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.
 
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.
 
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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.<br /><br />
 
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.<br /><br />
 
After double transformation, colony PCR was done and agarose gel shows that there remains the original DNA fragment.
 
After double transformation, colony PCR was done and agarose gel shows that there remains the original DNA fragment.
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Revision as of 01:28, 21 October 2019

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.
The Reverse Transcriptase Collection
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
The Reverse Transcription Initiation Collection
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 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
The Degradation Tag Collection
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).
The Cre Recombinase with Degradation Tags Collection
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)
The lox Sites Collection
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.