Contribution

This year, we're launching a cross-disciplinary project that integrates knowledge from various fields for better coordination. As we develop a compatible platform, we've designed and built modules that span from wet-lab to dry-lab, aimed at supporting future iGEM teams. Throughout this journey, we've encountered challenges and gained invaluable experience in addressing them. Our contributions include:

Parts: We have generated a novel semi-random mutagenesis tool, named as CBE-Mag-RNAP. This is a Cytosine Base Editor fused with T7 RNA polymerase which can do mutagenesis on specific DNA region between T7 promoter and T7 terminator. By incorporating blue light inducible p-Mag and n-Mag system, we are able to control the start and stop when using the CBE-Mag-RNAP mutagenesis system.

Improvement of Other Teams' Parts: We incorporated already existing parts, such as BBa_K4942007, which is a EGFP gene. This showed a new application of the gene as a sequence that can help evaluate the extent of mutagenesis, whether in the positive or negative direction in terms of fluorescence. This change can also demonstrate whether mutagenesis is on-going for other proteins as well.

New Proposal on Metabolic System Evolution: Here, we leverage the concepts from system biology and evolution. We were creating mutations on different enzymes at the same time in one metabolic system. A group set of mutations on different enzymes can make the system to be more productive.

Unbiased High-Throughput Screening Workflows: Here, we build a fluorescent based or a condition selection system by utilizing fluorescence-activated cell sorting (FACS) and next generation sequencing (NGS).

Potential Candidate Mutations Library for longevity: Here, we record several mutations which may contribute to anti-oxidation and anti-inflammation.

1.Parts

We tested 5 main scalable composite parts, developed in the Description part, tested in the Engineering and Results parts,and recorded in the Parts page.

Here, we have designed a tool that can induce controllable mutations in a specified segment of the genome without affecting other regions. The first part was created by fusing Cytosine Base Editor (CBE) with n-Mag and N-T7 RNA polymerase gene. The second part involved fusing p-Mag with C-T7 RNA polymerase. This blue-light-controllable system is designed to enable precise control over mutagenesis in a region extending from the T7 promoter to the T7 terminator, acting as a “start” and “stop” button for the process. By leveraging this technique, we were able to select and optimize specific metabolic pathways in Escherichia coli (E. coli), including identifying effective mutations to enhance the pentose phosphate pathway (PPP), accelerate the spermidine synthetic pathway, and increase the nicotine degradation pathway. All the selected mutations helped Caenorhabditis elegans achieve a longer lifespan and improved behavior. In results Figure 1 and Figure 2, we tested the blue-light inducible CBE-Mag-RNAP system. We have successfully created some mutations based on GFP gene tested by the Sanger sequencing (Result Figure 2).

2. Improvement of Previous Part BBa_K4942007

As included in our design for our "black box” protein, our team adapted the part BBa_K4942007 originally designed by iGEM23_SHSID-China, which was an Enhanced Green Fluorescent Protein (EGFP) gene. In our project, EGFP was included in our plasmid for the “black box” protein as a reporter gene, as the activation of mutagenesis can be easily detected via the fluorescence change in EGFP, which is much more convenient in evaluation compared to any other gene. In fluorescent microscopy and further quantification, we discovered the enhanced fluorescence in many of the EGFP proteins that have underwent mutagenesis. Moreover, using sequencing data to predict a mutant 3D protein, we provided rationale for the change in the fluorescence level. A newly added benzene ring likely benefited the congregation of these proteins. In addition, we found the use of EGFP to not only indicate whether mutagenesis is going on but also whether we are shining the system with enough blue light for enough mutagenesis, by evaluating the extent of the change in fluorescence. As a result, our team is able to ensure the adequate mutagenesis of many other enzymes that we aim to optimize in our project, including those involved in the synthesis of spermidine and NADPH as well as the degradation of nicotinate.

3. New Proposal on Metabolic System Evolution

In this project, we introduced three pathways into E. coli: the pentose phosphate pathway (PPP), the polyamine synthesis pathway (as shown in Figure 1A), and the nicotinate degradation pathway (as shown in Figure 1B). By activating the CBE-Mag-RNAP system, the base editor can simultaneously and randomly convert C to T in these genes without impacting the expression of other genes. We will select positive mutations that enhance NADPH and spermidine production for Sanger sequencing and next-generation sequencing (NGS). Additionally, mutations that improve nicotinate degradation will be identified using high concentrations of nicotinate. All surviving bacterial strains will undergo both NGS and Sanger sequencing to confirm the mutations.

Figure1. Three enhanced pathways in E. coli. A, The pentose phosphate pathway (PPP) and the polyamine synthesis pathway. B, The nicotinate degradation pathway.

4. Unbiased High-Throughput Screening Workflows

As shown in the Results section, we created a novel unbiased high-throughput screening workflow by measuring continuously expressed mCherry and GFP. In the process, we exploited the use of both fluorescence-activated cell sorting (FACS) and next generation sequencing (NGS). First by using FACS, we can discern fluorescence intensities of GFP and mCherry, and sorted out the cell populations with increased and decreased fluorescence intensities. Next, we uses next generation sequencing (NGS) to determine the mutated sequence that lead to the increase and decrease of fluorescent intensity. Measuring the overall intensity of different colonies, we could choose the most successful mutation by large scale sequencing. Specific condition based selection can be applied to test “gain of function” mutation with different enzymes. Upon implementing the unbiased high-throughput screening workflow to the resulting cell, we build a fluorescent based and condition based selection system.

Figure2. The condition selection system based on fluorescence-activated cell sorting (FACS) and next generation sequencing (NGS). A, Discerning cell populations with increased and decreased fluorescence intensities of GFP and mCherry by utilizing FACS. B, Determining the mutated sequence by utilizing NGS. C, The most successful GFP mutant (GFPT10I, S30F, A156V).

5. Potential Candidate Mutations Library for longevity

In the Results section, we proposed several mutations that enhances C. elegans lifespan, and some winner mutations could be documented as a universal reference. For example, the mutations registered as an improvement of the metabolic pathway of C. elegans in our experiment could be universal due to the resemblance of metabolic pathways in all organisms. In an innovative approach, we can document different function of mutations, contributing to a wide variety of uses including anti-oxidation, anti-inflammation and etc. The main purpose of tracing winner mutations is to code for a wide range of enhancement that later could be implemented in the eukaryotic cell for the benefit on a macro scale.

Figure3. Examples of mutations that enhances C. elegans lifespan. A, Structure based analysis of the winner mutation of spermidine synthase. B, Structure based analysis of the winner mutation of S-adenosylmethionine decarboxylase.

By establishing a high-throughput screening platform, we have been able to undertake directed evolution studies on genes sourced from animals and humans, conducting the mutagenesis and screening process in bacterial and yeast host systems. This has allowed us to systematically link specific mutations to functional enhancements, generating valuable data and insights. The knowledge gained from these efforts will provide important guidance and reference points for future gene therapy applications as well as the engineering of beneficial symbiotic microorganisms.

Figure4. System evolution by using CBE-Mag-RNAP system.