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<li class="leftNavLi"><a class="leftNavA" href="#mainTitle3">Recombination</a> | <li class="leftNavLi"><a class="leftNavA" href="#mainTitle3">Recombination</a> | ||
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<li class="leftNavLi2"><a class="leftNavA2" href="#mainTitle3_2">Cre initiates excision between two homologous loxP site</a></li> | <li class="leftNavLi2"><a class="leftNavA2" href="#mainTitle3_2">Cre initiates excision between two homologous loxP site</a></li> | ||
<li class="leftNavLi2"><a class="leftNavA2" href="#mainTitle3_3">lox5171 is most incompatible with wildtype loxP (wtlox)</a></li> | <li class="leftNavLi2"><a class="leftNavA2" href="#mainTitle3_3">lox5171 is most incompatible with wildtype loxP (wtlox)</a></li> | ||
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− | <li class="leftNavLi"><a class="leftNavA" href="#mainTitle4"> | + | <li class="leftNavLi"><a class="leftNavA" href="#mainTitle4">Modeling</a></li> |
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− | + | We testified experimentally that our separate modules could function well, while our modelling result proves for the theoretical applicability of our system design. | |
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+ | The mutagenesis system could be split into three modules, transcription, reverse transcription, and recombination. Meanwhile our modelling is also conducted in three modules, induced expression model, reverse transcription model and Cre recombination model. Experimental data are processed and proved by modelling result, mutually they testified and demonstrated the feasibility of our system. | ||
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+ | To ensure that our system can work safely and efficiently, we carefully stuck to all safety rules and investigated thoroughly to verify that each part is safe before using them. For more information please check our <a href="https://2019.igem.org/Team:Fudan-TSI/Safety">Safety</a> page. | ||
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− | We put the target sequence and its flanking elements together under a T7 stable promoter for high expression level of target RNA. To verify our R-Evolution system, we constructed | + | We put the target sequence and its flanking elements together under a T7 stable promoter for high expression level of target RNA. To verify our R-Evolution system, we constructed 8 nonsense mutant of chloramphenicol resistance gene (Chl), bearing the 8 base pair substitution from sense codon to nonsense mutant. We verified this construct through culturing bacteria carrying the original version or mutant on plates containing chloramphenicol. We found that bacteria carrying the original Chl grow naturally, while no colony was formed on the plates of mutated Chl (Fig. 1). |
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− | + | We also constructed and verified nonsense mutants of fluorescent protein EGFP and mCherry at the 158<sup>th</sup> and 159<sup>th</sup> amino acid (Fig. 2). | |
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− | + | We conducted SDS-PAGE on whole-cell lysates of uninduced and induced cell. Gag-pol polyprotein is expressed as a whole with its stop codon mutated into its readthrough product, glutamine. The polyprotein has three functional parts, capsid protein, protease and reverse transcriptase. We made Y586F mutation on reverse transcriptase to increase its mutation rate. From the PAGE gel, we can see that all three bands could be seen when induced (Fig. 3). | |
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− | <div class="col">Cre | + | |
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+ | <div class="col">Cre initiates excision between two homologous loxP site</div> | ||
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− | + | Placing 2 wild-type loxP on both ends of the target sequence (mCherry) in the same direction, and expressing it under a stable promoter (J23101). By co-transforming the target plasmid with another plasmid carrying Cre recombinase, we verified that our Cre protein functions accordingly by excising the mCherry sequence from the promoter (Fig. 4). Through PCR amplification with the primers annealing to sequences outside the target, and subsequent electrophoresis, we found that the band from bacteria co-transforming Cre corresponds to the excision of mCherry. | |
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− | <img src="https://static.igem.org/mediawiki/2019/ | + | <img src="https://static.igem.org/mediawiki/2019/e/e8/T--Fudan-TSI--Result_Fig5.gif" style="width:50%; margin:auto;"> |
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− | <b>Figure 4. | + | <b>Figure 4. lox511 remains compatible with wildtype loxP, though at a lower excision rate.</b><br /> |
− | + | Wildtype loxP and lox511-mCherry-loxP are analyzed on two different gels, their marker bands are indicated. Wildtype loxP only has an excision band. lox511 has a slight full-length mCherry band slightly longer than 1000 bp, which correlates with the full length between two loxP, but excision band is still visible and brighter than that of full-length mCherry. This result suggests that lox511 still interacts with wildtype loxP and go through excision, but at a lower efficiency. | |
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− | <img src="https://static.igem.org/mediawiki/2019/ | + | <img src="https://static.igem.org/mediawiki/2019/9/9e/T--Fudan-TSI--Result_Fig6.gif" style="width:60%; margin:auto;"> |
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<b>Figure 5. lox511 remains compatible with wildtype loxP, though at a lower excision rate. </b><br /> | <b>Figure 5. lox511 remains compatible with wildtype loxP, though at a lower excision rate. </b><br /> | ||
− | Wildtype loxP and lox511-mCherry-loxP are analyzed on two different gels, their marker bands are indicated. Wildtype loxP only has an excision band. lox511 has a slight full-length mCherry band slightly longer than 1000 bp, which corresponds with the | + | Wildtype loxP and lox511-mCherry-loxP are analyzed on two different gels, their marker bands are indicated. Wildtype loxP only has an excision band. lox511 has a slight full-length mCherry band slightly longer than 1000 bp, which corresponds with the Ctr+ result in Fig. 6, but excision band is still visible and brighter than that of full-length mCherry. This result suggests that lox511 still interacts with wildtype loxP and go through excision, but at a lower efficiency. |
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− | When we were carrying out integrated human practice, we were warned by Prof. Wang that two homologous loxP would be excised at a much higher efficiency than performing recombination as we wished, so we searched the literature and selected 3 mutants that are said to be incompatible with wt-loxP but are compatible with themselves, they are lox511, lox2272 and lox5171. We tested their incompatibility with wt-loxP by replacing one of wt-loxP into the mutant at the ends of mCherry, and co-transformed the plasmid with Cre (Fig. 5 & 7). The result we obtained showed that lox5171-mCherry-wt_loxP performs best, and used it in further analysis (Fig. 7). | + | When we were carrying out <a href="https://2019.igem.org/Team:Fudan-TSI/Human_Practices">integrated human practice</a>, we were warned by Prof. Wang that two homologous loxP would be excised at a much higher efficiency than performing recombination as we wished, so we searched the literature and selected 3 mutants that are said to be incompatible with wt-loxP but are compatible with themselves, they are lox511, lox2272 and lox5171. We tested their incompatibility with wt-loxP by replacing one of wt-loxP into the mutant at the ends of mCherry, and co-transformed the plasmid with Cre (Fig. 5 & 7). The result we obtained showed that lox5171-mCherry-wt_loxP performs best, and used it in further analysis (Fig. 7). |
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− | <b> | + | <b>Figure5. Schematic diagram of loxP mutant incompatibility test. </b><br /> |
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− | <b>Figure | + | <b>Figure 6. Cre excises sequences flanked by homologous loxP sites, but are incompatible with its mutant version. </b><br /> |
The above column shows which plasmids are transformed. The 3 middle lanes stand for Cre co-transforming with mCherry flanked on both ends by wildtype loxP (Lane 3), or with wildtype loxP on only one end, the other end being lox2272 (Lane 4) or lox5171 (Lane 5). mCherry flanked with lox2272 or lox5171 on one end does not go through excision so a full-length band was detectable, while mCherry flanked with wildtype loxP on both ends are excised and only a shorter band was seen. | The above column shows which plasmids are transformed. The 3 middle lanes stand for Cre co-transforming with mCherry flanked on both ends by wildtype loxP (Lane 3), or with wildtype loxP on only one end, the other end being lox2272 (Lane 4) or lox5171 (Lane 5). mCherry flanked with lox2272 or lox5171 on one end does not go through excision so a full-length band was detectable, while mCherry flanked with wildtype loxP on both ends are excised and only a shorter band was seen. | ||
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− | When our modelling demonstrated to us that the expression level of Cre needs to be much lower than that of RT, we introduced degradation tags. By attaching them to the C terminal of Cre recombinase, the protein would be rapidly recognized and degraded by the E. coli’s native SsrA-SmpB degradation system. This construct could also solve the problem of basal leakage and continued existence after inducer removal. Apart from the native AANDENYALAA tag, we also modified its last three or five amino acids into YALAV, YALVA, YALVV and WVLAA. We tested the stable expression level of each tag by attaching them to the C terminal of EGFP protein and measuring the change in fluorescence level (Fig. | + | When our modelling demonstrated to us that the expression level of Cre needs to be much lower than that of RT, we introduced degradation tags. By attaching them to the C terminal of Cre recombinase, the protein would be rapidly recognized and degraded by the E. coli’s native SsrA-SmpB degradation system. This construct could also solve the problem of basal leakage and continued existence after inducer removal. Apart from the native AANDENYALAA tag, we also modified its last three or five amino acids into YALAV, YALVA, YALVV and WVLAA. We tested the stable expression level, as well as the degradation dynamic of each tag by attaching them to the C terminal of EGFP protein and measuring the change in fluorescence level (Fig. 7). The stable state expression increases as the number of mutated amino acids increase, or the mutated site nears the N’ of the tag. Supported by our modelling result, we deemed that the WVLAA tag performs best and chose to use it in further experiments. |
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− | <b>Figure | + | <b>Figure 7. Degradation tag greatly reduces the protein level at stable state. </b><br /> |
WT represents the positive control of EGFP without any tag attachment. The five degradation tags are represented by their last five amino acid sequence. The vertical axis shows the quantitative analysis of EGFP fluorescence (excitation wavelength: 485 nm; detection wavelength: 528 nm), normalized by cell amount (OD600). The fluorescence is quantified by the concentration of green fluorescein, cell number is quantified by the number of silicon beads, both are from the distributed measurement kit. Fluorescence below detection level are eliminated. Error bar stands for the SEM of 3 replicates. t-test is performed between WT and each degradation tag, P<0.0001 (****). | WT represents the positive control of EGFP without any tag attachment. The five degradation tags are represented by their last five amino acid sequence. The vertical axis shows the quantitative analysis of EGFP fluorescence (excitation wavelength: 485 nm; detection wavelength: 528 nm), normalized by cell amount (OD600). The fluorescence is quantified by the concentration of green fluorescein, cell number is quantified by the number of silicon beads, both are from the distributed measurement kit. Fluorescence below detection level are eliminated. Error bar stands for the SEM of 3 replicates. t-test is performed between WT and each degradation tag, P<0.0001 (****). | ||
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− | + | Our modelling successfully demonstrated that our system could function and mutation could accumulate along bacteria growth (Fig. 8). For detailed explanation of our system, please visit our Modeling page. | |
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− | + | <img src="https://static.igem.org/mediawiki/2019/d/df/T--Fudan-TSI--DemoMd.gif" style="width:65%; margin:auto;"> | |
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+ | <b>Figure 8. Recombined Ptarget would occur when RT and Cre is expressed at a proper level. </b><br /> | ||
+ | Dynamics of the percentage of un-recombined/ recombined P<sub>target</sub> among all P<sub>target</sub>s is shown in the upper panel. The distribution of the percentage of substances at the steady-state is shown in the lower panel. Ps: un-recombined P<sub>target</sub>. Pp: recombined P<sub>target</sub>. The result that intermediate formed by un-recombined P<sub>target</sub> and T7RNA polymerase shows that mutation on P<sub>target</sub> can accumulate. | ||
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Revision as of 23:29, 21 October 2019
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