Difference between revisions of "Team:Fudan-TSI/Composite Part"
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| − | <p class="flow-text">For best composite part, we would like to present you with our <a href="/Team:Fudan-TSI/Part_Collection">Cre recombinase together with degradation tag</a>. Cre recombinase <a href="http://parts.igem.org/Part:BBa_K3257044" target="_blank">(BBa_K3257044)</a> is responsible for the recombination process between the reverse transcribed cDNA and the target | + | <p class="flow-text">For best composite part, we would like to present you with our <a href="/Team:Fudan-TSI/Part_Collection">Cre recombinase together with degradation tag</a>. Cre recombinase <a href="http://parts.igem.org/Part:BBa_K3257044" target="_blank">(BBa_K3257044)</a> is responsible for the recombination process between the reverse transcribed cDNA and the target gene, which is rather one of the most important processes in our system.</p> |
| − | <p class="flow-text">Because of the leakage of uninduced Cre expression, we have spent much effort in lowering the steady-state expression level of Cre. An important approach is to attach 5 different types of degradation tags after the CDS of Cre. Using EGFP <a href="http://parts.igem.org/Part:BBa_E0040" target="_blank">(BBa_E0040)</a> as a reporter, we can see that the degradation rate of degradation tag with amino acid residual sequence of AANDENWVLAA <a href="http://parts.igem.org/Part:BBa_K3257073" target="_blank">(BBa_K3257073)</a> is moderate for Cre recombinase to reach a steady but relatively low level of expression.</p> | + | <p class="flow-text">Because of the leakage of uninduced Cre expression, we have spent much effort in lowering the steady-state expression level of Cre. An important approach is to attach 5 different types of degradation tags after the CDS of Cre. Using EGFP <a href="http://parts.igem.org/Part:BBa_E0040" target="_blank">(BBa_E0040)</a> as a reporter, we can see that the degradation rate of degradation tag with amino acid residual sequence of AANDENWVLAA <a href="http://parts.igem.org/Part:BBa_K3257073" target="_blank">(BBa_K3257073)</a> is moderate for Cre recombinase to reach a steady but relatively low level of expression (Figure 1).</p> |
<p class="flow-text">Transformed into BL21(DE3) with another plasmid containing lox sites and analyzed by colony PCR, we can see that DNA cleavage between identical-oriented loxP sites happens indicating the normal function of Cre recombinase <a href="#Fig2">(Figure 2)</a>.</p> | <p class="flow-text">Transformed into BL21(DE3) with another plasmid containing lox sites and analyzed by colony PCR, we can see that DNA cleavage between identical-oriented loxP sites happens indicating the normal function of Cre recombinase <a href="#Fig2">(Figure 2)</a>.</p> | ||
Revision as of 17:03, 21 October 2019
Composite Parts
For best composite part, we would like to present you with our Cre recombinase together with degradation tag. Cre recombinase (BBa_K3257044) is responsible for the recombination process between the reverse transcribed cDNA and the target gene, which is rather one of the most important processes in our system.
Because of the leakage of uninduced Cre expression, we have spent much effort in lowering the steady-state expression level of Cre. An important approach is to attach 5 different types of degradation tags after the CDS of Cre. Using EGFP (BBa_E0040) as a reporter, we can see that the degradation rate of degradation tag with amino acid residual sequence of AANDENWVLAA (BBa_K3257073) is moderate for Cre recombinase to reach a steady but relatively low level of expression (Figure 1).
Transformed into BL21(DE3) with another plasmid containing lox sites and analyzed by colony PCR, we can see that DNA cleavage between identical-oriented loxP sites happens indicating the normal function of Cre recombinase (Figure 2).
Figure 2. The verification of DNA cleavage between LoxP sites.
Plasmids containing the Cre gene were co-transformed with plasmids containing mCherry flanked by LoxP sites. A pair of sequencing primers were used to amplify the mCherry gene. PCR product would be around 1000 bp while fragments cleaved by Cre recombinase would be approximately 250 bp. The positive control group is the PCR product of plasmids containing the original mCherry gene. The negative control group is the PCR product of plasmids containing the Cre gene only.
For more information of other composite parts, please refer to the part collection page and the iGEM Part Registry ranging from BBa_K3257000 to BBa_K3257076.
Project by Team:Fudan-TSI
Mutation library generation is critical for biological and medical research, but current methods cannot mutate a specific sequence continuously without manual intervention. We hereby present a toolbox for in vivo continuous mutation library construction. First, the target DNA is transcribed into RNA. Next, our reverse transcriptase (RT) reverts RNA into cDNA, during which the target is randomly mutated by our RT's enhanced error-prone ability. Finally, the mutated version replaces the original sequence through recombination. These steps will be carried out iteratively, generating a random mutation library of the target with high efficiency as mutations accumulate along with bacterial growth. Our toolbox is orthogonal and provides a wide range of applications among various species. R-Evolution could mutate coding sequences and regulatory sequences, which enables the evolution of individual proteins or multiple targets at a time, promotes high-throughput research, and serves as a foundational advance to synthetic biology.