<figcaption><i>Fig 7: Fluorescence (eCFP) was observed only for bacterial cells harbouring either plasmids C2 or C3, and induced with IPTG. Within each frame, from left to right, -ve control, 0.5mM IPTG, 1.0mM IPTG. <b>[LEFT]</b> Triplicates from fast induction. <b>[RIGHT]</b> Triplicates from slow induction.</i></figcaption>
<figcaption><i>Fig 6: Fluorescence (eCFP) was observed only for bacterial cells harbouring either plasmids C2 or C3, and induced with IPTG. Within each frame, from left to right, -ve control, 0.5mM IPTG, 1.0mM IPTG. <b>[LEFT]</b> Triplicates from fast induction. <b>[RIGHT]</b> Triplicates from slow induction.</i></figcaption>
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<p>Following the qualitative analysis of our reporter systems, a quantitative analysis of our reporter systems was performed to further corroborate their functionallity. A similar IPTG induction protocol was followed, where bacterial cultures were induced with IPTG and incubated at 37°C for 2 hours. Our quantitative results corroborate our qualitative analysis, where only fluorescence was observed in IPTG-induced BL21 (DE3) cells harbouring plasmid C2 or C3.
<figcaption><i>Fig 8: Quantification of relative fluorescence intensity for all three reporter systems. From left to right, C1(-IPTG), C1(+IPTG), C2(-IPTG), C2(+IPTG), C3(-IPTG) and C3(+IPTG) <b>[LEFT]</b> Replicate 1; n=8 technical replicates. <b>[CENTER]</b> Replicate 2; n=6 technical replicates. <b>[RIGHT]</b> Replicate 3; n=6 technical replicates.</i></figcaption>
<figcaption><i>Fig 7: Quantification of relative fluorescence intensity for all three reporter systems. From left to right, C1(-IPTG), C1(+IPTG), C2(-IPTG), C2(+IPTG), C3(-IPTG) and C3(+IPTG) <b>[LEFT]</b> Replicate 1; n=8 technical replicates. <b>[CENTER]</b> Replicate 2; n=6 technical replicates. <b>[RIGHT]</b> Replicate 3; n=6 technical replicates.</i></figcaption>
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<p>Having robustly evaluating our reporter systems through both qualitative and quantitative methods, we eventually decided on plasmid C3 as our final reporter plasmid. This was mainly based on two reasons:
<figcaption><i>Fig 9: Inferred ancestral tree for both datasets. Box in red in wild-type T7RNAP sequence. <b>[LEFT]</b> Inferred ancestral tree generated from Blast100; boxed in blue are nodes of interests. Nodes 119 (denoted as RNAPAnc119) and 137 (denoted as RNAPAnc137) were selected for further investigation. <b>[RIGHT]</b> Inferred ancestral tree generated from Blast250; circled in red are nodes of interests. Node 302 (denoted as RNAPAnc302) was selected for further investigation.</i></figcaption>
<figcaption><i>Fig 8: Inferred ancestral tree for both datasets. Box in red in wild-type T7RNAP sequence. <b>[LEFT]</b> Inferred ancestral tree generated from Blast100; boxed in blue are nodes of interests. Nodes 119 (denoted as RNAPAnc119) and 137 (denoted as RNAPAnc137) were selected for further investigation. <b>[RIGHT]</b> Inferred ancestral tree generated from Blast250; circled in red are nodes of interests. Node 302 (denoted as RNAPAnc302) was selected for further investigation.</i></figcaption>
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<p><listyle="font-weight: bold;">Structure Prediction using Alphafold</li>
<spanstyle="display: inline-block;"> Alphafold was then utilized to first predict the structure of all ancestral sequences of interest, providing insights into the protein's folding and functional domains. PyMOL was also employed to visualize the predicted structure, allowing for a detailed comparison with the wild-type and the RMSD value. After confirming the feasiblity of the structure through computational analyses, the ancestral sequences were then cloned into our stable T7-expressing plasmid, plasmid 1c (see <ahref="https://2024.igem.wiki/ntu-singapore/engineering">Engineering Success</a> for details), for downstream testing and analysis.</span>
<figcaption><i>Fig 10: Predicted structure for ancestral sequences of interest using Alphafold. <b>[TOP]</b> Alphafold predicted structures for all 3 ancestral sequences; and compared against the structure of wild-type T7RNAP (cyan). <b>[BOTTOM LEFT]</b> Sequence coverage of RNAPAnc119. <b>[BOTTOM CENTER]</b> Sequence coverage of RNAPAnc137. <b>[BOTTOM RIGHT]</b> Sequence coverage of RNAPAnc302. </i></figcaption></div></p>
<figcaption><i>Fig 9: Predicted structure for ancestral sequences of interest using Alphafold. <b>[TOP]</b> Alphafold predicted structures for all 3 ancestral sequences; and compared against the structure of wild-type T7RNAP (cyan). <b>[BOTTOM LEFT]</b> Sequence coverage of RNAPAnc119. <b>[BOTTOM CENTER]</b> Sequence coverage of RNAPAnc137. <b>[BOTTOM RIGHT]</b> Sequence coverage of RNAPAnc302. </i></figcaption></div></p>
<p>The ancestral sequences were subsequently cloned into plasmid 1c using Gibson assembly, replacing the wild-type T7RNAP sequence. Purified plasmids were then transformed into competent <i>E. coli</i> reporter cells to compare their efficiency against the wild-type for an initial screening. Cells were then sub-cultured, followed by IPTG induction. Comparing the different variables tested, it is evident that the wild-type T7RNAP expressed from a plasmid (Stbl3 - 1c/C3) has shown the highest relative fluorescence intensity among all, higher than that observed by BL21 (DE3) cells transformed with the reporter plasmid. Comparing both the ancestral sequences (RNAPAnc119 and RNAPAnc137), little to no fluorescence intensity was similar, showing that the derived ancestral sequences have much lower processivity than the wild-type. As such, more developmental effots to improve the processitivity of the ancestral sequences are required before a comparable efficiency is observed. The level of fluorescence intensity observed from that of the uninduced negative control at 120mins is also higher than that of some of the induced samples. As the sequence of the antisense oligonucleotide (ASO) was designed specifically for the wild-type T7RNAP, it is possbily that the ASO are not acting against the ancestral sequences. This results in the ancestral sequences to not be supressed by the ASO system. Hence, future works also include developing and refining the sequence of the ASO for different ancestral sequences.
<figcaption><i>Figure 10: Comparison of fluorescence measured at different time point after IPTG induction (-ve(120min,10min, 30min, 60min, 120min). 6 technical measurements were taken for each sample. The negative control sample were measured after the 120 minutes timepoint. </i></figcaption>
<figcaption><i>Fig 10: Comparison of fluorescence measured at different time point after IPTG induction (-ve(120min,10min, 30min, 60min, 120min). 6 technical measurements were taken for each sample. The negative control sample were measured after the 120 minutes timepoint. </i></figcaption>
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<p>Several of the mutations were cloned into T7RNAP individually, and evaluated for their efficiency after IPTG-induction. The variation of the fluorescence measured between each construct is high. At timepoint 10min, the fluorescence intensity for most variants are negative, with fluorescence increasing with time (higher fluorescence intensity measured at later timepoints of 30min and 60min).
<li>The fluorescence intenisty measured for constructs a - e, and g are relatively similar, although with slight fluctuations.</li>
<li>The fluorescence intenisty measured for construct h, i , k, and m are exceptionally low compared to the entire panel tested.
<li>The fluorescence intenisty measured for construct J appear to be the highest amongst the variant constructs; ableit still lower than the wild-type T7RNAP.</li></p>
<li>The fluorescence intenisty measured for construct J appear to be the highest amongst the variant constructs; ableit still lower than the wild-type T7RNAP.</li>
<li>Additional established variants will be added to the panel to further include more beneficial mutations.</li>
<figcaption><i>Figure 11: Comparison of different constructs with site directed mutagenesis at different time point after IPTG-induction (10min, 30min, 60min, 120min). 6 technical replicates were taken for each variant. The negative control sample were measured after the 120 minutes timepoint.</i></figcaption></div></p>
<figcaption><i>Fig 11: Comparison of different constructs with site directed mutagenesis at different time point after IPTG-induction (10min, 30min, 60min, 120min). 6 technical replicates were taken for each variant. The negative control sample were measured after the 120 minutes timepoint.</i></figcaption></div></p>
<p>Another method that can be utilized to identify beneficial mutations is through screening mutants after random mutageneis. Random mutagenesis is a widely utilized technique in molecular biology and directed evolution that generates genetic diversity by introducing random mutations into DNA sequences. The various methods to induce random mutagenesis include error-prone PCR (epPCR), chemical mutagenesis, transposon-based methods, saturation mutagenesis and mutator strains<sup>6</sup>.</p>
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<p>We thus employed epPCR to generate a library of mutant T7RNAPs, in which the wild-type T7RNAP sequence was amplified using a low-fidelity DNA polymerase that introduces point mutations during amplification. The resulting PCR products, which contain a library of mutations, were then cloned into plasmid 1c, replacing the wild-type sequence. Sanger sequencing results from few colonies show random point mutations along the T7RNAP sequence (not shown). The library of mutant plasmids was then transformed into our competent <i>E. coli</i> reporter cells and plated onto CmR + Kan LB Agar plates.
<figcaption><i>Figure 12: Library of mutant T7RNAP plasmids transformed into reporter E. coli cells</i></figcaption></div></p>
<figcaption><i>Fig 12: Library of mutant T7RNAP plasmids transformed into reporter E. coli cells</i></figcaption></div></p>
<p> The cells were collected and diluted 100x, before aliquoting 125ul each into a 96 well plate for further sub-culture. However, wells along the 96-well plate were completely dried up, with partial drying of culture observed throughout the plates. As such, the identification of mutant variants was unable to proceed due to in-optimization of experimental protocols.
<figcaption><i>Figure 13: Observed dried up cultures along the edges of the 96-well plates, resulting in auto-fluorescence when observed under blue light.</i></figcaption></div></p>
<figcaption><i>Fig 13: Observed dried up cultures along the edges of the 96-well plates, resulting in auto-fluorescence when observed under blue light.</i></figcaption></div></p>
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<p>One of the final experiments performed was based on one of the feedback questions received during our public engagement efforts (see <ahref="https://2024.igem.wiki/ntu-singapore/human-practices">Human Practices</a>). One of the questions we received prompted us to determine if our current expression system (the combination of plasmid 1c with C3) was capable of incorporating pseudouridines into mRNA transcripts, or if the addition of pseudouridines would impair the catalytic function of the T7 RNA polymerase. Bacterial cultures containing the various T7-expression systems were induced with IPTG w/o pseudouridines. The lack of significant differences in the levels of fluorescence detected between the same expression system suggests that pseudouridine does not have a substantial effect on the experimental setup. This indicates that the presence of pseudouridine may not influence the reporter's sensitivity or the overall outcome of the experiment, leading to the conclusion that it might not be a critical factor in this particular context. Further investigation may be necessary to explore its role or to confirm these findings under different conditions.
<figcaption><i>Figure 14: Samples were induced with IPTG w/o pseudouridines to determine the effect of pseudouridines on expression levels.</i></figcaption></div></p>
<figcaption><i>Fig 14: Samples were induced with IPTG w/o pseudouridines to determine the effect of pseudouridines on expression levels.</i></figcaption></div></p>
<p>Given the incorporation of pseudouridines in the production of mRNA vaccines, the ability to incorporate such uncanonical bases would prove to be useful for such applications. Future experiments such as using in-vitro transcription with modified bases could thus be performed to select for variants that are capble of incorporating these unnatural bases.</p>
