Difference between revisions of "Team:Fudan-TSI/Demonstrate"
| Line 2,117: | Line 2,117: | ||
<div class="row"> | <div class="row"> | ||
<div class="col"> | <div class="col"> | ||
| − | 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. | + | 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 <a href="https://2019.igem.org/Team:Fudan-TSI/Model"><u>Modeling</u></a> page. |
</div> | </div> | ||
Revision as of 03:38, 22 October 2019
We testified experimentally that our separate modules could function well, while our modelling result proves for the theoretical applicability of our system design.
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.
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 Safety page.
Transcription
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 7 nonsense mutant of chloramphenicol resistance gene (Chl), bearing the 7 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).
Figure 1. The flanking of reverse transcription initiation and loxP sequences does not disrupt Chl functionality.
Wildtype Chl gene continues to express and colonies are grown on the plate containing chloramphenicol (plate on the right). The L158X mutant cannot express functional resistance gene and thus the plate is devoid of cell colony (plate on the right).
Wildtype Chl gene continues to express and colonies are grown on the plate containing chloramphenicol (plate on the right). The L158X mutant cannot express functional resistance gene and thus the plate is devoid of cell colony (plate on the right).
We also constructed and verified nonsense mutants of fluorescent protein EGFP and mCherry at the 158th and 159th amino acid (Fig. 2).
Figure 2. Nonsense mutant of EGFP and mCherry cannot emit fluorescence.
The vertical axis shows the quantified level of EGFP expression. Mutants of the fluorescence proteins cannot emit fluorescence while the wildtype protein functions naturally. The fluorescence level (EGFP: excitation wavelength 485 nm, detection wavelength 528 nm; mCherry: excitation 550 nm, detection 590 nm) is quantified by the concentration of fluorescein, and normalized by the measured OD600 equivalent to the number of beads in the system. The fluorescein for EGFP and silica beads are from the iGEM distributed measurement kit, red fluorescence is quantified by rhodamine B (Sigma-Aldrich, #S1402-1G). Error bar in the two graphs on the first row indicates the SEM of three replicates.
The vertical axis shows the quantified level of EGFP expression. Mutants of the fluorescence proteins cannot emit fluorescence while the wildtype protein functions naturally. The fluorescence level (EGFP: excitation wavelength 485 nm, detection wavelength 528 nm; mCherry: excitation 550 nm, detection 590 nm) is quantified by the concentration of fluorescein, and normalized by the measured OD600 equivalent to the number of beads in the system. The fluorescein for EGFP and silica beads are from the iGEM distributed measurement kit, red fluorescence is quantified by rhodamine B (Sigma-Aldrich, #S1402-1G). Error bar in the two graphs on the first row indicates the SEM of three replicates.
Reverse transcription
The successful expression of reverse transcriptase
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).
Figure 3. gag-pol polyprotein is successfully expressed and underwent excision in the cell.
SDS-PAGE is performed on whole cell-lysis. The gag-pol polyprotein is split into three pieces, capsid protein (60.4 kDa), protease (13.5 kDa) and reverse transcriptase (69.1 kDa). Both versions of reverse transcriptase, one wildtype, the other Y586F mutant, are tested. ‘-’ stands for uninduced sample, while ‘+’ stands for sample after induction. From the gel we could see that all three bands are brighter in the induced sample.
SDS-PAGE is performed on whole cell-lysis. The gag-pol polyprotein is split into three pieces, capsid protein (60.4 kDa), protease (13.5 kDa) and reverse transcriptase (69.1 kDa). Both versions of reverse transcriptase, one wildtype, the other Y586F mutant, are tested. ‘-’ stands for uninduced sample, while ‘+’ stands for sample after induction. From the gel we could see that all three bands are brighter in the induced sample.
Recombination
Cre initiates excision between two homologous loxP site
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.
Figure 4. lox511 remains compatible with wildtype loxP, though at a lower excision rate.
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.
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.
lox5171 is most incompatible with wildtype loxP (wtlox)
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).
Figure5. Schematic diagram of loxP mutant incompatibility test.
Figure 6. Cre excises sequences flanked by homologous loxP sites, but are incompatible with its mutant version.
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.
Cre with degradation tags
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.
Figure 7. Degradation tag greatly reduces the protein level at stable state.
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 (****).
Modeling
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.
Figure 8. Recombined Ptarget would occur when RT and Cre is expressed at a proper level.
Dynamics of the percentage of un-recombined / recombined Ptarget among all Ptargets 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 Ptarget. Pp: recombined Ptarget. The result that intermediate formed by un-recombined Ptarget and T7RNA polymerase shows that mutation on Parget can accumulate.
Dynamics of the percentage of un-recombined / recombined Ptarget among all Ptargets 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 Ptarget. Pp: recombined Ptarget. The result that intermediate formed by un-recombined Ptarget and T7RNA polymerase shows that mutation on Parget can accumulate.