Project Description

Outline:
1. Background:why we do this project? 2. Two Adjusted Editions for System Improvement 3. Three strategies for Applications 4. Four Key Dry Lab Skill 5. Safety 6. User manual 7. Conclusion

1. Background:why we do this project?

Mutations are alterations in the genetic sequence and are a key driver of diversity among organisms. These changes happen at various levels and can lead to a wide range of outcomes. What makes this fascinating is that genetic diversity enables different types of interactions between organisms, such as symbiosis, predation, competition, and parasitism. Together, these interactions contribute to what we call an "ecosystem."

Mutagenesis is the process of altering an organism's DNA, leading to mutations that change its genetic information. Many physical and chemical mutagenesis techniques have been used to create "improved" plants. For instance, larger strawberries and sweeter corn are products of beneficial mutations.

While mutagenesis offers exciting possibilities for improving the environment for humans, there is impossible when applying physical and chemical mutagenesis to higher organisms. Because these methods are random and uncontrollable, they can result in deformities or even fatal effects in animals and humans.

How to design/obtain a better gene functioning in animals or human to avoid disease and aging??? There is no idea we can get from current literatures.

To achieve this idea by enhancing some of their genes, we first choose those genes from higher organism and put them downstream of a T7 promoter in the engineered bacteria or yeast.

Next, we performed semi-random mutagenesis using Cytosine Base Editor- Mag-T7 RNA polymerase system (CBE-Mag-RNAP). While adding IPTG to bacteria to start expressing CBE-nMag-nRNAP and pMag-cRNAP, we get the mutagenesis system ready to use. When performing the blue light, the RNAP can be assembled and start to work on the genes downstream of T7 promoter. We call it the black box system because this CBE-Mag-RNAP can generate a semi-random mutation on Cytosine in a region starting from T7 promoter to T7 terminator.

In order to know how many mutations contributed to an optimized system, we next need to design a selection strategy.

1) A fluorescent based high-throughput screen strategy.

We first incorporate a GFP report system. This system can report how the base editor functioning in the cells.

2) A fluorescent and nutrition selection based high-throughput screen strategy.

By leveraging GFP as a base editor reporter, we need to select out those bacteria which can be better survived in the selection LB plate. So we chose two direction to do the mutagenesis and selection.

(2.1) Enhancing metabolic products by mutating key enzymes in pentose phosphate pathway (PPP) and spermidine synthesis pathway in Escherichia coli (E. coli).

(2.2) Enhancing the nicotine (mimic by nicotinate) degradation by mutating key enzymes in nicotinate degradation pathway in E. coli.

Additionally, we used the same techniques as previously with the second generation black box for enzymes in the nicotinate degradation pathway with nicotinate solution applied to the LB plate. We've recorded mutations that improve nicotinate degradation, which could contribute to air purification efforts.

The exciting results keep coming out and we are so excited about it!

2.Two Adjusted Editions for System Improvement

(1) First generation

Our first generation of Black box is inspired from a Nature Communication Journal[1] by Dr. Aaron Cravens and his team, which first designed a new in-vivo mutagenesis in which the base editing protein was attached and guided by the RNA polymerase.

In the first generation of Black box system, there are highly distinctive T7 promoter, which allows T7 RNAP to slide down with the CBE in the specific area only and mutate some C to T randomly. Also, we got the LacO to control the start of the process.

Though we solve the broad-spectrum limitation problem, there are some shortcomings with the design. Mainly, we can't stop this process and all C will be mutated into T in the end, leading to only one result.

(2) Second generation

Are there any mechanisms or tools that allow us to better control the base editor?A Nature chemical biology journal[2] inspired us. Dr. Jinyue Pu and her team reported a development of a proximity-dependent split T7 RNA polymerase. Another article by Dr. Sara Dionisi[3] showed that the split of T7 RNA polymerase can be controlled by blue light. Therefore, using the idea of blue light controlled interactions between fused proteins modulating the assembly of a split RNAP, we designed a second generation Cytosine Base Editor (CBE) Black Box System that is more controllable. We introduced a T7 RNA polymerase that consists of two protein subunits, each linked to their respective photoswitch—pmag and nmag—that integrates the two subunits upon blue light activation. The cytosine base editor is attached to one of the subunits. Once assembled, the polymerase can then begin transcription starting at the T7 promoter site. Besides introducing T7 RNA polymerase, the new system also features green fluorescent protein that would break down upon the initiation of transcription, which functions as an indicator to see if the system is working properly. As the protein complex (CBE) is guided through the DNA sequence by the T7 RNA polymerase, it performs efficient, precise, and even random (given the blue light is switched on and off randomly) mutagenesis.

3. Three strategies for Applications

We have received attention for the system we designed that reveals the essence of occurrences. Some groups andorganizationsgot in touch with us to ask questions and to show interests in applying this technique to address some of their issues. By analysing the changes in fluorescence intensity of fluorescent proteins and sequencing results, we have verified the success of the second generation black box system and intend to apply it to three different objects (environmental, human, fungal).We use the system to increase the activity of several key enzymes in those areas, which we test in nematodes.

(1) Purifying environments by breaking down nicotine

It is well-established that second-hand smoke exerts detrimental effects on the environment and local ecosystems. By engineering targeted mutations that enhance bacterial nicotine degradation, we can potentially develop a bioproduct to mitigate these negative impacts.

Our process began by leveraging the KEGG database to identify key enzymes involved in the E. coli nicotine metabolic pathway, including nicotinate dehydrogenase, 6-hydroxynicotinate reductase, and enamidase. We then combined second-generation plasmids with nicotine plasmids introduced them into BL21 strains for mutations. We also combined the generation plasmid with the nicotine plasmid and mutated it in the sensory state as a control.

We then introduced the mutated E. coli into a nicotine-enriched environment using nematodes as a biological model, while maintaining a control group which we fed nematodes with unmutated BL21 for comparison. The exercise per unit time and the survival rateof the nematodes served as an indicator of the bacteria’s enhanced nicotine degradation ability, with the mutated strains showing significant improvements in nicotine breakdown. This led to the development of a novel bioproduct containing the mutated bacteria, designed to reduce the harmful effects of second-hand smoke by effectively absorbing nicotine from contaminated environments.

(2) Increase lifespan by improving spermidine synthesis

Scientists have long discovered that increasing spermidine levels in organisms helps delay aging. By recording some mutations related to enhanced spermidine synthesis and incorporating them into the E. coli metabolic system, we can develop a product for intestinal probiotics.

Similar to our work in nicotine degradation, we first examined the spermidine synthesis pathway in E. coli, identified the key enzymes, and then applied our Black Box system for mutation and selection.

We validated the proper functioning of our system through sequencing. Next, we fed nematodes with both non-mutated and mutated E. coli respectively and placed them in two petri dishes containing arginine for cultivation as a control. Additionally, we set up a positive control group treated with spermidine to demonstrate the rigor of our experiment. The results showed that the nematodes fed with the mutated E. coli had a longer average lifespan than those fed with the non-mutated E. coli, and their lifespan was very close to that of the positive control group, proving the success of our experiment.

With this, we can proceed to process the mutated bacteria into edible probiotics, allowing us to produce high-quality biological products.

(3) Prevent fungi invasion by developing bacteria to oxidative stress

Same as what we have done in degrading nicotine, we first examined the pentose phosphate pathway in E. coli, identified the key enzymes, and then applied our Black Box system for mutation and selection. Similarly, through sequencing, we validated the proper functioning of our system. Next, we fed nematodes with both mutated BL21 containing plasmids and regular BL21, placing them in petri dishes containing hydrogen peroxide for cultivation. After some time, we observed that the nematodes fed with the mutated BL21 had a significantly longer lifespan than those fed with the regular strain, demonstrating the success of our experiment. This confirmed that the mutated BL21 can produce more of the antioxidant NADPH and can reduce oxidative stress. We are now working to apply this mutation to treat cobweb mold disease in morel mushrooms.

4. Four Key Dry Lab Skills

(1) Plasmid construction

Plasmid code for various enzymes in a metabolic system. To demonstrate one’s project in plasmid is to include substantial evidence, so by constructing a viable and successful plasmid one can show the both the practicality and the implementable value of a system.

Three examples of construction of plasmid could demonstrate this:

First, we constructed a spermidine metabolic plasmid, coding for the various enzymes in the spermidine to arginine pathway. We coded the essential enzymes S-adenosylmethionine and decarboxylase-RBS-agmatinase inside the plasmid that could be responsive for a system that implement gene editing in the sequence, making the pathway more productive. This in turn shows the value of plasmid construction

Next, we constructed a energy metabolic pathway, specializing in the production of more NADPH. By exploiting the system on various sequence that encodes vitial enzymes in the energy pathway, we saw a great increase in cellular growth inside the host. This show the importance of creating a successful plasmid for demonstration of your project.

Last, we provide the cell with a nicotine degradation pathway, targeting the objective to degrade nicotine which is toxic to cellular activity. By utilizing control over enzyme coding sequences, alternating essential enzymes by our project, the demonstration of the viability of our implementation to the cell. Therefore, the integration of plasmid construction is important for project feasibility and practical study.

Overall, construction of plasmid for implementation of the system could be an essential way for project well being as it provided substantial evidence for the project’s effect.

(2) DNA Sequencing and Analysis

We submit our samples and turn them over to a synthesis company that sequences them for us. The principle is to determine the composition of DNA by reading its base sequence. For example, we use sequencing as a technology in our project to determine whether our results are successful. First, the genomic sequence of the sample is obtained by high-throughput sequencing technology, and then it is aligned with a reference genome to identify mutation sites. This mutation information is then collated and entered into a dictionary to form a mutation database for subsequent analysis and functional validation. This process not only improves the efficiency of mutation detection, but also enables in-depth study of genomic variants.

In our project, we use sequencing technology, when the sequencing results appear in two bumps in one paragraph at the same time, it means that one letter of the gene sequence is mutated to another, indicating that our results have been mutated. If the sequencing results of arginine and nicotine are successful, it means that the black box lipid particles can work properly in both environments. In nicotine sequencing, we judge each cleavage site and the number of cleavage sites to determine if it meets our expectations. Once we have determined whether the results were successful, sequencing was used to record those more obvious mutations that were enhanced. We then use these mutations to describe our dictionary, which records the enhancement mutations and hopefully enhances the metabolism of bacteria and enhances the yield of biological products. Let the species that originally have this function strengthen their own functions, and let some species that do not have this function buy to achieve this function, and the success of the sequencing results allows us to make predictions about the mutation of organisms.

(3) Structural prediction

Within our dry lab portion, another crucial part is the prediction of structure, something that accounts for all of the function in regards to a molecule. Luckily, computer softwares, in the past few years, have grown to develop capabilities in analyzing structures of molecules, such as proteins. AlphaFold is an in-silico tool that has recently been developed by Google DeepMind to utilizes the principle of machine learning to facilitate protein prediction. It is trained with information on homologous structures and multiple sequence alignment to do so. Here, we use the newest version of the software that was released in 2024, AlphaFold 3 (accessible via https://alphafoldserver.com/),to predict the structure of proteins involved in our project.

We predicted the structure of our “black box” protein. Especially for the 2nd edition of the protein where proteins n-mag and p-mag allow the splitting and connection of two different sub proteins, AlphaFold 3 predicted the individual sub proteins as well as the potential interaction between the two. From the structure simulation, we can observe the correct binding between the p-mag and n-mag components to form a functioning “black box” for mutagenesis, which provides confidence that our protein can function in mutating the targeted DNA in vitro.

In addition, all of the proteins that has underwent our wet lab procedure for mutagenesis were analyzed with protein prediction via AlphaFold. Specifically, the sequence retrieved from Sanger Sequencing were submitted to the computer software. For the Green Fluorescent Protein that was designed to analyze the effects of our protein, our in-silico analysis of its structure aimed to explain its change in fluorescence levels, which depended on how the structure could potentially impact the congregation of numerous GFPs to increase the intensity of fluorescence. Moreover, AlphaFold has the capabilities in revealing the rationale for the improvement of the various enzymes that we aim to optimize. The structure of the active cleft is largely responsible for the changes in the protein’s catabolic interactions with its ligands, for which we also analyzed and developed conclusions from.

(4) Metabolic pathways

The reason for our introduction of the reference of KEGG in our project is to guide our plasmid construction. Our goal is to use controlled mutagenesis to exploit a organism for a specified target, and the current problem is about the practical effect of this system, so we aim to use our system to mutate pre-exist functional genes in order for the demonstration of our project. KEGG demonstrates the classic metabolic pathway, and it show each step and enzyme to transform a bio molecule to another(accessible via https://www.kegg.jp/kegg/kegg1.html). So by analyzing different KEGG pathways, we can implement our system effectively.

We find two ways that we could apply our system in KEGG. The first is about making the project more feasible. In the original plan of exploiting the system for the degradation of nicotine, we find that it is illegal to exploit pure nicotine, so we trace the KEGG pathway and find a substitute: nicotinate. The metabolic web helps us to maneuver around difficulty in the design.

Next, KEGG could provide valuable insights to the formulation of experiments. In the metabolic process involving transformation of spermidine to arginine, we encountered a problem of differentiating product arginine. However, after long time research about the chemical orientation present in the KEGG metabolic pathway, we found a novel use of Phenolphthalein indicator in determining the product. With a clear and well-informed pathway, we could demonstrate our project more feasibly.

Overall, the KEGG metabolic pathway could help us to demonstrate mutagenesis. By referencing to the KEGG map, we can formulate eligible experiments and find alternative designs with current problems. It is evident both in the practical implementation of controllable random mutagenesis in the transformation of spermidine to arginine and the degradation of nicotine.

5. Safety

(1) Arabinose is found in the natural environment. Once arabinose reacts in the environment, Cas9 will start gene editing, which is harmful and can lead to the death of bacteria.

(2) In the lab, arabinose can be dropped on harmful bacteria to kill them, which can limit the cells from running out of the lab and have an impact on a wider area. This can kill cells in the laboratory, which can facilitate the effective blocking of living things.

6. User manual

We have designed these bio-products to be easy to use in the appropriate scenarios, and here are some pictures shows how to use them.

By taking a high-quality biological product containing mutant bacteria, it helps the body produce more spermidine, which has the effect of extending life.

Sprinkling the plant with a mutated fungus with stronger antioxidant properties inhibits the growth of the fungus that causes white mould, thus making it possible to cure white mould in morel mushrooms.

By combining activated carbon with our mutated bacteria, which are more efficient at degrading nicotine, we have created an activated carbon bottle capable of absorbing nicotine. Simply placing it in areas with smoke can effectively reduce nicotine levels in the environment. After some time, the carbon bottle can be reactivated by leaving it in the sunlight, allowing it to be reused.

7. Conclusion

By addressing the difficulty of causing advantageous mutations without endangering the organism, these initiatives enable safer and faster directed evolution. The platform we have established can help to develop new biological products and solve some specific problems, which is a two-way protection for people and the environment, which is also precisely the original intention of our project.

Reference:
[1]Cravens, A., Jamil, O. K., Kong, D., Sockolosky, J. T. & Smolke, C. D. Polymerase-guided base editing enables in vivo mutagenesis and rapid protein engineering. Nature Communications 12, (2021).
[2]Pu, J., Zinkus-Boltz, J. & Dickinson, B. C. Evolution of a split RNA polymerase as a versatile biosensor platform. Nature Chemical Biology 13, 432–438 (2017).
[3]Dionisi, S., Piera, K., Baumschlager, A. & Khammash, M. Implementation of a novel optogenetic tool in mammalian cells based on a split T7 RNA polymerase. ACS Synthetic Biology 11, 2650–2661 (2022).