iGEM 2024 — Team ntu-singapore

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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Attached is the account of our lab work progress in a chronological order.

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
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## Introduction

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The iGEM Engineering Cycle composes of 4 stages – **Design**, **Build**, **Test** and **Learn** – each entailing a developmental process towards an ideal biological system. The main processes behind each stage in the iterative process is as follows:

- **Design** – Conceptualization and planning; brainstorming possible ideas
- **Build** – Build our biological system through molecular cloning techniques
- **Test** – Test our biological system and evaluate its efficiency
- **Learn** – Reflect on its flaws and improve the system

This iterative process allows us to refine our biological system through the mistakes learnt from the previous cycle, thereby building towards an ideal biological system.

![](https://static.igem.wiki/teams/5401/engine-page/engine-1.png)

*Fig 1: iGEM Engineering Cycle*

## Design-Build-Test-Learn

Our iGEM Engineering Cycle encompasses the developmental process behind our T7RNAP-expressing plasmid. Below shows the 3 iterations, each detailing the **features**, **flaws** and **improvements** to the construction of our plasmid. The aim of the Engineering Cycle was to build a plasmid that was capable of expressing T7 RNA Polymerase (T7RNAP) stably, thereby allowing us to investigate our protein of interest.

---

Iteration I - Plasmid 1 [Click to expand]

Design - Expression of T7RNAP under a constitutive promoter J23104.
- High-copy number plasmid (pUC) with a strong origin of replication (oriR).
- Chloramphenicol as the selection marker.

Build

The plasmid was constructed using Gibson assembly involving parts BBa\_J364008 (as the backbone) and BBa\_J435007 (encoding T7RNAP).

![](https://static.igem.wiki/teams/5401/engine-page/engine-2.png)

*Fig 2: Design of our initial T7-expressing plasmid - plasmid 1*

Test

Despite 2 separate rounds of cloning the desired plasmid, we were unable to isolate our plasmid of interest with the correct sequence.- In the first round of cloning, our T7 expression cassette was missing several expression elements (ie. J23104 promoter, RBS and terminator) to properly express the T7RNAP. The defects observed in these clones were despite the fact that the primers used and original plasmids have already contained these elements.

![](https://static.igem.wiki/teams/5401/engine-page/engine1-1.png)
![](https://static.igem.wiki/teams/5401/engine-page/engine1-2.png)

*Fig 3: Identified missing expression elements in Plasmid 1 after Sanger sequencing. **[LEFT]** Missing constitutive promoter J23104 and RBS. **[RIGHT]** Missing rrnB T1 terminator and T7Te terminator*

- In the second round of cloning, new primers were designed and utilised for cloning. However, we still observed either a truncated protein or a deletion of part of the T7RNAP sequence in several of the colonies sequences.

![](https://static.igem.wiki/teams/5401/engine-page/engine1-3.png)
![](https://static.igem.wiki/teams/5401/engine-page/engine1-4.png)

*Fig 4: Identified missing protein sequences in Plasmid 1 after Sanger sequencing. **[LEFT]** Missing 'T' in the first start codon ATG, resulting in the expression of a truncated protein variant. Whilst not shown, the truncated variant was still capable of transcription. **[RIGHT]** Deletion of the entire T7RNAP coding sequence; resulting in only the immediate N- and C-terminus amino acid residues*

Learn

The defects observed from both rounds of cloning seems to predominantly lie with the issue of stably expressing the T7 RNAP within its host; which ties in which the issue of expression toxicity and lethality threshold.

Conventionally, T7RNAP is used in *in vivo* protein expression (eg. BL21 (DE3) or other DE3 counterparts) owing to its specific promoter recognition and high transcriptional activity1. In these strains, T7RNAP is commonly expressed under an inducible promoter, to regulate the expression and allow the temporal control of T7RNAP (ie. induce only when required). This temporal control of T7RNAP has several benefits, with the most obvious linking to the fitness of the host.

T7RNAP is reported to have 8-fold higher transcriptional activity than host bacterial RNAPs2. As such, when cloning the T7RNAP under a constitutive promoter, we hypothesis the high transcriptional activity of T7RNAP to eventually become toxic to its host, When such a strong RNA polymerase is constitutively expressed in a host, it lowers the fitness of the host due to inefficient allocation of metabolic resources.
This forces the host to negatively select variants through undesirable mutations (usually with worse-performing traits) to better counteract the original strong RNA polymerase activity3. This was what we observed where the cloning of T7RNAP under a constitutive promoter had several setbacks.

As such, to better regulate the expression of T7RNAP and mitigate the toxicity associated, we designed the constitutive promoter to be under the regulation of a lac operon, a system commonly used in commercial DE3 bacterial strains.

Iteration II - Plasmid 1a [Click to expand]

Design - Expression of T7RNAP under a regulated promoter; constitutive promoter J23104 is upstream of and regulated by a lac operator.
- Addition of lacI repressor to aid in the regulation of the promoter-lac operator.

Build

The plasmid was constructed using Gibson assembly involving parts BBa\_J364008 (as the backbone) and BBa\_J435007 (T7RNAP). LacI sequence with promoter was derived from plasmid Addgene #65767.

![](https://static.igem.wiki/teams/5401/engine-page/engine-3.png)

*Fig 5: Design of our second T7-expressing plasmid - plasmid 1a*

Test

The cloning of plasmid 1a was relatively easier, with the various expression elements and the sequence of the protein still intact. The addition of regulatory elements shows promise in regulating the expression of T7RNAP. The plasmid (plasmid 1a) was subsequently transformed into competent *E. coli* reporter cells already containing our reporter plasmid, and plated onto double antibiotic (Kan + CmR) LB agar plates. Colony PCR was then performed to determine if both plasmids were retained within the *E. coli* reporter cells. Whilst the colony PCR results show that both plasmids were retained, it also showed an increase in the band size of the fragment corresponding to the T7RNAP, possibly due to an insertion sequence.

![](https://static.igem.wiki/teams/5401/engine-page/engine2-1.png)
![](https://static.igem.wiki/teams/5401/engine-page/engine2-2.png)

*Fig 6: Colony PCR results after transforming plasmid 1a into competent E.coli reporter cells. **[LEFT]** Whilst the band of interest (expected size: ~800bp) corresponding to our reporter casette in plasmid C3 was correct, the band of interest corresponding (observed to be either ~2000bp or ~3000bp) to plasmid 1a was observed to be different from the expected size of 1200bp. **[RIGHT]** Colony PCR was repeated using untransformed plasmids as control. Only the band of interest derived from the control samples showed the expected band size of 1200bp.*

Transformation was thus performed again, with plating onto two different antibiotic agar plates, as well as the addition of a control. Colony PCR was also performed again to observe for any similar pattern of insertion sequences, and if the insertion was harboured near the N-terminus or C-terminus of the T7RNAP. Our results support the previous observation where there was an insertion sequence within the open reading frame of the T7RNAP coding sequence, with insertion sequence found nearer to the N-terminus. The frequency of these insertion sequences was also observed to be higher in our reporter cells.- [1] - Plasmid 1a transformed into competent *E. coli* reporter cells and plated onto Kan + CmR LB agar plates
- [2] - Plasmid 1a transformed into competent *E. coli* reporter cells and plated onto CmR LB agar plates
- [3] - Plasmid 1a transformed into competent empty *E. coli* cells and plated onto CmR LB agar plates

![](https://static.igem.wiki/teams/5401/engine-page/engine2-3.png)

*Fig 7: Colony PCR was performed again after transformation of plasmid 1a into competent *E. coli* reporter and empty cells. An increase in band size was observed in the fragment corresponding to the T7RNAP sequence. The colony PCR results showed that the insertion sequence is consistently found closer to the N-terminus coding region of the protein. Box in red are colonies that were further cultured for plasmid purification.*

In order to determine the identity of the insertion sequences, colonies that were boxed in red were further cultured to perform plasmid purification (miniprep). Plasmids were isolated and sent for Sanger sequencing to reveal the identity of these insertion sequences. Sanger sequencing results from [2]-4 support the observation that the increase in band size was due to an insertion sequence (identified as IS1 by Snapgene) within the coding region (near the N-terminus of T7RNAP). The location of the insertion was also highly specific, between amino acids 315 and 317.

![](https://static.igem.wiki/teams/5401/engine-page/engine2-4.png)

*Fig 8: Sanger sequencing results of plasmid 1a purified from [2]-4. The insertion sequence was identified as IS1, and was inserted before amino acid positions 315 - 317.*

Learn

The insertion sequence (IS1) within the coding sequence of T7RNAP was hypothesized to be a "defence mechanism" from the Stbl3 *E.coli* cells after transformation. This observation was also more commonly observed when transformed with competent cells already containing the reporter plasmid. It was possible that the IS1 sequence acts to “break” the coding sequence of T7RNAP, thereby knocking out the expression of the T7RNAP. Overall, while the plasmid was clone-able, our test results suggest that the expression of T7RNAP from plasmid 1a is possibly still toxic and unstable despite the additional regulatory elements such as the lac operator (lacO) and lacI repressor. This suggests that the current regulatory elements are still insufficient to control the expression of T7RNAP.

To further regulate and maintain a stable expression of T7RNAP, an antisense oligonucleotide (ASO) system and a lower-copy number plasmid system was adopted – plasmid 1c.

Iteration III - Plasmid 1c [Click to expand]

Design - Addition of an antisense oligonucleotide (ASO) with Hfq scaffold sequence to promote the knockdown of T7RNAP expression4. The ASO aims to targets the +4 to +27 of the coding sequence to knockdown the expression of T7RNAP.
- Medium-copy number plasmid using a pBR322 backbone

Build

The plasmid was built on from plasmid 1a; using Gibson assembly to add in the antisense oligonucleotide (ASO) system and swap the oriR. ASO was ordered as a gBlock and different oriR was derived from BBa\_J428341 (medium-copy pBR322 plasmid).

![](https://static.igem.wiki/teams/5401/engine-page/engine-4.png)

*Fig 9: Design of our third T7-expressing plasmid - plasmid 1c*

Test

The cloning of plasmid 1c was swiftly completed, with its sequence verified. Previous regulatory elements (ie. lacO and lacI) were retained, and the ASO system was also included. Similarly, plasmid 1c was subsequently transformed into our competent *E. coli* reporter cells for an initial screening. Transformed cells were then plated across three different LB agar plates - CmR only, Kan only, and CmR + Kan.

![](https://static.igem.wiki/teams/5401/engine-page/engine3-1.png)

*Fig 10: Colonies were observed for all three different LB agar plates after transformation.*

8 colonies from each plate were then selected for colony PCR; to determine if the respective plasmids were retained within the *E. coli* cells. Colony PCR results indicate that both the T7-expressing plasmid (CmR resistance marker) and reporter plasmid (Kan resistance marker) were retained on all 8 colonies on the double antibiotic plate (CmR + Kan). These colonies were inoculated and further subcultured. Fluorescence was subsequently observed in only colony 6 after a period of incubation (initially grown at 37°C overnight; then kept at 4°C for 72 hours).

![](https://static.igem.wiki/teams/5401/engine-page/engine3-2.png)

*Fig 11: 8 colonies per plate were selected for colony PCR. Colonies derived from plates on single antibiotic only contained a single plasmid (their corresponding markers). Colonies derived from double antibiotic plate (CmR + Kan) contained both plasmids, albeit with different fragment sizes for the T7RNAP (different sizes are possibly due to deletion or insertion of sequences within the coding region of T7RNAp; however, this was not further pursued due to the interest of time).*

![](https://static.igem.wiki/teams/5401/engine-page/engine3-3.png)

*Fig 12: Fluorescence was observed in only colony 6 after bacterial cells were pelleted after incubation. Initial results suggest the reporter system is functional, and that T7RNAP can be stably expressed. Observation of fluorescence is also in line with colony PCR results. **[TOP/a]** Observation under blue-light. **[BOTTOM/b]** Observation under physical light.*

In order to determine if the expression of T7RNAP from plasmid 1c is indeed more stable than that of plasmid 1a, we transformed our *E. coli* reporter cells again with respective plasmids, and performed colony PCR to identify for the presence of any possible insertion sequences within the coding sequence of the T7RNAP. The same pair of primers were used to amplifiy the sequence of interest for both plasmids.- Expected band size for plasmid 1a: 1892bp
- Expected band size for plasmid 1c: 2038bp; due to additional ASO system

![](https://static.igem.wiki/teams/5401/engine-page/engine3-4.png)

*Fig 13: Colony PCR results showed the presence of insertion sequences within the coding sequence of T7RNAP for E.coli reporter cells transformed with plasmid 1a. Band size corresponding to ~3000bp (different from expected band size of 1892bp) was observed for all 8 colonies picked from reporter cells transformed with plasmid 1a, thereby showing the unstable expression of T7RNAP.*

Learn and Further Actions

Through our Engineering Cycle, we were able to refine the expression of T7RNAP, a protein that was initially unstable. A combination of various regulatory elements work hand-in-hand together, aimed at knocking the expression levels of T7RNAP down to stable levels. Our last iteration also capitalizes on an ASO system, a system that is not commonly used for plasmid-expressed proteins. Overall, the utilisation of the different regulatory elements allowed us to stably express the wild-type T7RNAP, thereby allowing us to investigate the efficiency of other variants in our random mutagenesis library.

In addition, given the scope of our project to also explore ancestral sequences, the ASO system can be further refined to regulate the expression of these T7RNAP variants that have low sequence homology.

## References

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1 CHAMBERLIN, M., MCGRATH, J., & WASKELL, L. (1970). New RNA Polymerase from Escherichia coli infected with Bacteriophage T7. *Nature (London)*, **228(5268)**, 227–231. <https://doi.org/10.1038/228227a0>

2 Miroux, B., & Walker, J. E. (1996). Over-production of Proteins inEscherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular Proteins at High Levels. *Journal of Molecular Biology*, **260(3)**, 289–298. <https://doi.org/10.1006/jmbi.1996.0399>

3 Kar, S., & Ellington, A. D. (2018). Construction of synthetic T7 RNA polymerase expression systems. , **143**, 110–120. <https://doi.org/10.1016/j.ymeth.2018.02.022>

4 Liang, X., Li, C., Wang, W., & Li, Q. (2018). Integrating T7 RNA Polymerase and Its Cognate Transcriptional Units for a Host-Independent and Stable Expression System in Single Plasmid. *ACS Synthetic Biology*, **7(5)**, 1424–1435. <https://doi.org/10.1021/acssynbio.8b00055>

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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Attached is the list of protocols performed for our experiments.

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{% block title %}Results{% endblock %}
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## Overview

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Our results consist of mainly 4 parts:- Firstly, developing and determining the right reporter system for evaluation.
- Secondly, evaluating the efficiency of WT-T7RNAP against derived ancestral sequences.
- Thirdly, evaluating the efficiecny of WT-T7RNAP against literature-reviewed variants.
- Lastly, evaluating the efficiency of WT-T7RNAP with addition of psuedouridine.

### Developing the Right Reporter System

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The very first part of our experiment was to design a reporter system to evaluate the efficiency of the wild-type T7RNAP, as well as any variants generated in the future. Established protocols that have previously characterized T7RNAP have utilized antibiotic resistance as a readout1, with better-performing variants capable of surviving at higher concentrations of antibiotic conditions2.

To prevent manipulating the fitness phenotype of the organism, our design primarily revolves around utilizing the expression of fluorescence as a reporter to evaluate the transcriptional activity of different T7RNAP variants. Our initial design was a simple reporter cassette, a cyan fluorescent protein encoded downstream of a T7 promoter. This reporter cassette was then ligated to a high-copy number (pUC) Kanamycin resistance plasmid.

The plasmid was constructed via ligation of different parts derived from the iGEM registry to construct the full reporter cassette and plasmid.- BBa\_J428353 - pJUMP28-1A(sfGFP)
- BBa\_K3457003 - T7 Promoter
- BBa\_J428032 - Ribosomal Binding Site (RBS)
- BBa\_E0020 - eCFP
- BBa\_J428091 - T7Te

![](https://static.igem.wiki/teams/5401/results-page/res1-1.png)

*Fig 1: Reporter cassette used for evaluating T7RNAP*

![](https://static.igem.wiki/teams/5401/results-page/c-t7promoter-ecfp-final-map.png)

*Fig 2: Reporter plasmid - plasmid C1 (pUC; high-copy number plasmid)*

The reporter plasmid was then transformed into BL21 (DE3) cells for testing. Colony PCR was first performed to confirm successful plasmid transformation before a single colony was inoculated and cultured overnight.The overnight culture was then sub-cultured at a 1:50 dilution for 3 hours until the OD600 reached approximately 0.400, after which, it was induced with 0.5 mM IPTG for 2 hours. Fluorescence was observed only in the IPTG-induced BL21 (DE3) cells, confirming that the reporter plasmid was functional.

![](https://static.igem.wiki/teams/5401/results-page/res1-2.png)

![](https://static.igem.wiki/teams/5401/results-page/res1-3.png)
![](https://static.igem.wiki/teams/5401/results-page/res1-4.png)

*Fig 3: Initial screening of our reporter plasmid C1. **[TOP]** Colony PCR confirmed successful transformation of plasmid C1 into BL21 (DE3) cells for all 8 colonies. Only 1 colony was inoculated and further cultured overnight. **[LEFT]** Observation of fluorescence under physical light. **[RIGHT]** Observation of fluorescence under blue-light.*

Unfortunately, upon further sub-culturing of the bacterial culture (-ve control) followed by repeated IPTG induction, no fluorescence was observed. We initially hypothesized the loss of fluorescence due to metabolic burden, caused by the expression of our protein of interest from a high-copy plasmid, resulting in a change in sequence within the reporter cassette. However, the plasmids isolated from the un-induced cultures showed no mutations within the T7 promoter sequence, nor within the coding sequence of the fluorescent protein.

To further investigate the loss of fluorescence previously observed in reporter C1 (BL21 (DE3) ells transformed with plasmid C1), Sanger sequencing was employed to identify for possible loss-of-function mutations in the coding regions of the T7RNAP found within the genome. The T7RNAP coding sequence nested within the genome of BL21(DE3) cells was first amplified using PCR, then purified and sent for Sanger sequencing. Sanger sequencing results revealed a mutation in the active site of the T7RNAP3 - I810T. It was possible that this change in amino acid residue in the active site of T7RNAP resulted in the loss of function of the enzyme, thereby resulting in the loss of fluorescence observed subsequently.

![](https://static.igem.wiki/teams/5401/results-page/res1-5.png)
![](https://static.igem.wiki/teams/5401/results-page/res1-9.png)

*Fig 4: Isolated plasmids from two un-induced samples and amplified T7RNAP sequence from the genome of BL21 (DE3) cells were sent for Sanger sequencing. **[TOP]** Sequencing results showed that the reporter cassette of plasmid C1 remains unchanged, with the features still intact in the same position. Sequence alignment was also performed to verify that there were no major changes in the nucleotide sequence (not shown here). **[BOTTOM]** Sanger sequencing identified a mutation within the active site of T7RNAP, I810T. This could have resulted in the loss of function of function of the enzyme, thereby resulting in the loss of fluorescence observed in subsquent sub-cultures.*

Due to the unstable expression of the fluorescence observed, two additional reporter plasmids were cloned using Gibson assembly. The reporter cassette was cloned into a medium-copy number plasmid (BBa\_J428341) and a low-copy number plasmid (BBa\_J428350) to provide a more stable reporter system.- Plasmid C2 – pBR322\_T7Prom\_eCFP – Medium-copy plasmid (15 – 20 copies per cell).
- Plasmid C3 - p15A\_T7Prom\_eCFP – Low-copy plasmid (10 copies per cell)

![](https://static.igem.wiki/teams/5401/results-page/c2-pbbr322-t7prom-ecfp-map.png)
![](https://static.igem.wiki/teams/5401/results-page/c3-p15a-t7prom-ecfp-map.png)

*Fig 5: Plasmid map for plasmid C1 and C2. **[LEFT]** Medium-copy plasmid (BBa\_J428341; 15 – 20 copies per cell). **[RIGHT]** Low-copy plasmid (BBa\_J428350; 10 copies per cell)*

Similarly, the reporter plasmids (plasmid C2 and C3) were transformed into BL21 (DE3) bacterial cells for testing. Colony PCR was performed (results not shown) to confirm successful transformation before a single colony was inoculated for further culturing. Following the culturing, all three plasmids (C1, C2 and C3) were evaluated for their functionality as a reporter plasmid in BL21 (DE3) *E. coli* cells. Overnight cultures of BL21 (DE3) *E. coli* cells containing the three plasmids respectively were first sub-cultured (at 1:25 dilution) for 2-3 hours before IPTG induction. Two different IPTG-induction protocols were followed:- Fast induction: Incubate at 37 °C for 2 hours after IPTG induction.
- Slow induction: Incubate at 18 °C for overnight after IPTG induction.

Qualitatively, across both IPTG-induction methods, the observations were consistent where fluorescence was only observed in BL21 (DE3) cells containing either reporter plasmids C2 or C3, and when they were induced with either 0.5mM or 1.0mM IPTG.

![](https://static.igem.wiki/teams/5401/results-page/res1-7.png)
![](https://static.igem.wiki/teams/5401/results-page/res1-8.png)

*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. **[LEFT]** Triplicates from fast induction. **[RIGHT]** Triplicates from slow induction.*

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.

![](https://static.igem.wiki/teams/5401/results-page/plasmidc1-ecfp-barchart.png)
![](https://static.igem.wiki/teams/5401/results-page/plasmidc2-ecfp-barchart.png)
![](https://static.igem.wiki/teams/5401/results-page/plasmidc3-ecfp-barchart.png)

*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) **[LEFT]** Replicate 1; n=8 technical replicates. **[CENTER]** Replicate 2; n=6 technical replicates. **[RIGHT]** Replicate 3; n=6 technical replicates.*

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:- By having our reporter cassette encoded on a low-copy number plasmid, we are able to increase the sensitivity of our assay, thereby allowing any difference in the fluorescence intensity to be easily detected. This aids in identifying better-performing variants in our downstream experiments.
- The utilisation of a plasmid with a p15A origin of replication also allows for the compatibility with our T7-expressing plasmid, which has a different origin of replication (see [Engineering Success](https://2024.igem.wiki/ntu-singapore/engineering)). As such, by using plasmids from different incompatibility groups for our experiments, we can prevent a competition of cell machinery and resources between the two plasmids.

### Evaluating the efficiency of WT-T7RNAP against derived ancestral sequences

---

Ancestral sequence reconstruction has a been a long-standing technique in evolutionary biology to infer sequences of ancient proteins based on existing sequences4. One of the key takeaways from our interviews (see [Human Practices](https://2024.igem.wiki/ntu-singapore/human-practices)) was the difficulty behind developing novel protein sequences through protein engineering. As such, we set out to use ancestral reconstruction to generate functional distant homologs through *in silico* methods. A programme called Molecular Evolutionary Genetics Analysis version X (MEGAX)5 was utilized for our ancestral reconstruction workflow:

- Data Procurement
Two datasets were generated by using the wild-type T7RNAP sequence as the query to search for homologous sequences using BLASTP (Basic Local Alignment Search Tool Protein). The first dataset generated ~100 sequences (denoted Blast100), all derived within the same taxonomy of Studiervirinae (tax id: 2731653). A larger dataset of ~250 (denoted Blast250) sequences was also derived with no limitations for a more diverse and possibly biologically relevant dataset.

- Sequence Alignment
The protein sequences within each dataset were then aligned and distant sequences were manually removed. Sequences that resulted in large gaps for all other sequences were subsequently removed to "reduce” these misalignment between sequences.

- Model Selection
An initial substitution model was ran to find the best model for ancestral reconstruction for each dataset. The LG model (G+I) was used for Blast100, while the LG model (G+I+F) was used for Blast250.

- Phylogenetic Tree Construction and Ancestral State Reconstruction
A maximum likelihood tree (phylogenetic tree) was then generated based on the best-fit model of each dataset. MEGAX was then utilized to infer the ancestral sequence based on the constructed phylogenetic tree and selected outgroup.

![](https://static.igem.wiki/teams/5401/results-page/studiervirinae4.png)

![](https://static.igem.wiki/teams/5401/results-page/inferred-ancestral-tree-blast250.png)

*Fig 8: Inferred ancestral tree for both datasets. Box in red in wild-type T7RNAP sequence. **[LEFT]** 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. **[RIGHT]** Inferred ancestral tree generated from Blast250; circled in red are nodes of interests. Node 302 (denoted as RNAPAnc302) was selected for further investigation.*

- Structure Prediction using Alphafold
 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 [Engineering Success](https://2024.igem.wiki/ntu-singapore/engineering) for details), for downstream testing and analysis.

![](https://static.igem.wiki/teams/5401/results-page/combined.png)

![](https://static.igem.wiki/teams/5401/results-page/studiervirinae42rnapanc119-c7abb-coverage.png)
![](https://static.igem.wiki/teams/5401/results-page/studiervirinaernapanc137-coverage.png)
![](https://static.igem.wiki/teams/5401/results-page/blastt7302-coverage.png)

*Fig 9: Predicted structure for ancestral sequences of interest using Alphafold. **[TOP]** Alphafold predicted structures for all 3 ancestral sequences; and compared against the structure of wild-type T7RNAP (cyan). **[BOTTOM LEFT]** Sequence coverage of RNAPAnc119. **[BOTTOM CENTER]** Sequence coverage of RNAPAnc137. **[BOTTOM RIGHT]** Sequence coverage of RNAPAnc302.*

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 *E. coli* 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.

![](https://static.igem.wiki/teams/5401/results-page/labelled-anc-0926-copy.jpeg)

*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.*

### Evaluating the efficiecny of WT-T7RNAP against literature-reviewed variants

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#### Site-Directed Mutagenesis

The project also aims to evaluate a series of mutations derived from literature, focusing on their functional implications to the T7RNAP. Below is a table summarizing the specific mutations along with their corresponding references. There are also future efforts to combine these mutations found across different literatures to developing better-performing variants.

| Code | Mutation | Reference |
| --- | --- | --- |
| A | S43Y | Wu, H., Wei, T., Yu, B., Cheng, R., Huang, F., Lu, X., Yan, Y., Wang, X., Liu, C., & Zhu, B. (2021). A single mutation attenuates both the transcription termination and RNA-dependent RNA polymerase activity of T7 RNA polymerase. RNA Biology, 18(sup1), 451–466. <https://doi.org/10.1080/15476286.2021.1954808> |
| B | G47A | Dousis, A., Ravichandran, K., Hobert, E. M., Moore, M. J., & Rabideau, A. E. (2022). An engineered T7 RNA polymerase that produces mrna free of immunostimulatory byproducts. Nature Biotechnology, 41(4), 560–568. <https://doi.org/10.1038/s41587-022-01525-6> |
| K | +884G |
| C | P266L | Guillerez, J., Lopez, P. J., Proux, F., Launay, H., & Dreyfus, M. (2005). A mutation in T7 RNA polymerase that facilitates promoter clearance. Proceedings of the National Academy of Sciences, 102(17), 5958–5963. <https://doi.org/10.1073/pnas.0407141102> |
| D | S430P | Boulain, J.-C., Dassa, J., Mesta, L., Savatier, A., Costa, N., Muller, B. H., L’hostis, G., Stura, E. A., Troesch, A., & Ducancel, F. (2013). Mutants with higher stability and specific activity from a single thermosensitive variant of T7 RNA polymerase. Protein Engineering Design and Selection, 26(11), 725–734. <https://doi.org/10.1093/protein/gzt040> |
| F | S767G |
| I | F880Y |
| M | I810S |
| G | F849I |
| L | Q744R |
| E | E643G |
| K | T3M | Jiang, K., Yan, Z., Bernardo, M. D., Sgrizzi, S. R., Villiger, L., Kayabolen, A., Kim, B., Carscadden, J. K., Hiraizumi, M., Nishimasu, H., Gootenberg, J. S., & Abudayyeh, O. O. (2024). Rapid Protein Evolution by Few-Shot Learning with a Protein Language Model. <https://doi.org/10.1101/2024.07.17.604015> |
| H | F880A | Gardner, L. P., Mookhtiar, K. A., & Coleman, J. E. (1997). Initiation, elongation, and processivity of carboxyl-terminal mutants of T7 RNA polymerase. Biochemistry, 36(10), 2908–2918. <https://doi.org/10.1021/bi962397i> |

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).- The fluorescence intenisty measured for constructs a - e, and g are relatively similar, although with slight fluctuations.
- The fluorescence intenisty measured for construct h, i , k, and m are exceptionally low compared to the entire panel tested.- The fluorescence intenisty measured for construct J appear to be the highest amongst the variant constructs; ableit still lower than the wild-type T7RNAP.
  - Additional established variants will be added to the panel to further include more beneficial mutations.

  ![](https://static.igem.wiki/teams/5401/results-page/labelled-snp-copy.jpeg)

  *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.*

#### Random Mutagenesis

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 strains6.

By generating large libraries of mutants, we can screen for variants with improved properties or novel functionalities, such as enhanced enzyme activity of stability under different conditions.

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 *E. coli* reporter cells and plated onto CmR + Kan LB Agar plates.

![](https://static.igem.wiki/teams/5401/results-page/photo-2024-10-02-19-30-29.jpg)

*Fig 12: Library of mutant T7RNAP plasmids transformed into reporter E. coli cells*

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.

![](https://static.igem.wiki/teams/5401/results-page/random-muta.jpg)

*Fig 13: Observed dried up cultures along the edges of the 96-well plates, resulting in auto-fluorescence when observed under blue light.*

### Evaluating the efficiency of WT-T7RNAP with addition of psuedouridine

---

One of the final experiments performed was based on one of the feedback questions received during our public engagement efforts (see [Human Practices](https://2024.igem.wiki/ntu-singapore/human-practices)). 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.

![](https://static.igem.wiki/teams/5401/results-page/pseudoutp-copy.png)

*Fig 14: Samples were induced with IPTG w/o pseudouridines to determine the effect of pseudouridines on expression levels.*

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 capable of incorporating these unnatural bases.

### References

---

1 Ikeda, R. A., Ligman, C. M., & Warshamana, S. (1992). T7 promoter contacts essential for promoter activity in vivo. *Nucleic acids research*, **20(10)**, 2517–2524. <https://doi.org/10.1093/nar/20.10.2517>

2 Boulain, J. C., Dassa, J., Mesta, L., Savatier, A., Costa, N., Muller, B. H., L'hostis, G., Stura, E. A., Troesch, A., & Ducancel, F. (2013). Mutants with higher stability and specific activity from a single thermosensitive variant of T7 RNA polymerase. *Protein engineering, design & selection: PEDS*, **26(11)**, 725–734. <https://doi.org/10.1093/protein/gzt040>

3 Bonner, G., Patra, D., Lafer, E. M., & Sousa, R. (1992). Mutations in T7 RNA polymerase that support the proposal for a common polymerase active site structure. *The EMBO Journal*, **11(10)**, 3767–3775. <https://doi.org/10.1002/j.1460-2075.1992.tb05462.x>

4 Joy, J. B., Liang, R. H., McCloskey, R. M., Nguyen, T., & Poon, A. F. Y. (2016). Ancestral Reconstruction. *PLoS Computational Biology*, **12(7)**, e1004763–e1004763. <https://doi.org/10.1371/journal.pcbi.1004763>

5 Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. *Molecular Biology and Evolution*, **35(6)**, 1547–1549. <https://doi.org/10.1093/molbev/msy096>

6 Labrou N. E. (2010). Random mutagenesis methods for in vitro directed enzyme evolution. *Current protein & peptide science*, **11(1)**, 91–100. <https://doi.org/10.2174/138920310790274617>

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