Engineering Success
1. Cycle 1-Building Controllable Semi-Random Mutagenesis System
(1)Design
The development of random mutagenesis is primarily motivated by the goal of addressing large-scale challenges. Random mutagenesis can either enhance or reduce the expression of genetically coded products. By applying random mutagenesis, selecting beneficial mutations, and refining the final product, we can identify the most effective mutation for various applications, ultimately helping to solve a wide range of problems.
We designed a more controllable mutagenesis second generation. The new system consists of the Lac operon, CBE (cytidine deaminase), and a functional T7 RNA polymerase. This system is capable of mutating cytosine to thymine from the T7 promoter to the terminator. However, due to its uncontrollable nature-where CBE mutates all cytosines in its path to thymine-the first-generation system was not suitable as a universal solution for problem-solving.
(2)Build
Seeing the shortcoming of the first-generation mutagenesis system, we seek to gain greater control over the entire random mutagenesis process. Building on previous experience, we have incorporated light-sensitive components-such as N-terminal RNAP, C-terminal RNAP, n-Mag, and p-Mag-into the existing T7 RNAP system. In this new, blue-light-sensitive T7 RNAP, exposure to blue light triggers the components to bind together, forming a functional RNAP that initiates transcription. By controlling the timing of transcription, we can more effectively select the gene that we hope to induce mutation on.
(3)Test & Learn
We performed semi-random mutagenesis using Cytosine Base Editor- Mag-T7 RNA polymerase system (CBE-Mag-RNAP). After collecting the data, we fit it and compared it with the model. 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.
Figure1. The Second Generation of controllable mutagenesis system
2. Cycle 2-High Throughput Screening Selection over Human Selection
(1)Design
The system is encoded into a plasmid for transformation, and initially, human selection is used to identify the most successful host cell by observing fluorescence under a microscope and picking the most prominent monoclonal colony. However, this technique is inefficient for large-scale selection. To address this, we turned to high-throughput screening, which allows for the effective selection of large numbers of cells.
(2)Build
We chose GFP as an indicator of successful mutation. In this approach, cells are placed in a harsh and selective environment to enhance plasmid production. For example, genetically modified Estrecholia coli(E. coli) can produce large amounts of plasmid in a petri dish containing over 20% glycerol and ampicillin. The host cells are then processed through the second-generation system and analysed using flow cytometry to select cells with the lowest fluorescence intensity. By Altering the GFP sequence entirely, it can indicate a positive mutagenesis of other enzyme coding parts in the plasmid. By high throughput screening techniques, we can select the most successful host cell in large quantities.
(3)Test & Learn
Figure2. Results for High Throughput screening selection for flourescent intensity
3. Cycle 3-Development of a monitor to quantify the process mutagenesis
(1)Design
We used metabolic genes instead of GFP as a parameter. We first incorporate a GFP report system. This system can report how the base editor functions in the cells.
(2)Build
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.
(3)Test & Learn
We are surprised to find some mutation can make the GFP brighter after we a high-throughput screening process using fluorescence- activated cell sorting (FACS) combined with Next Generation Sequencing. Mutations that enhance GFP brightness, known as "Winner Mutations", are cataloged in our Dictionary System.
4. Cycle 4 - Improvement from GFP to metabolic system evolution using CBE-Mag-RNAP
(1)Design
After confirming the second-generation of black box could successfully enhance the frequency of random mutations, we applied it to improve the pentose phosphate pathway (PPP), polyamine synthesis pathway and nicotine metabolic pathway in E. coli. We hypothesized that by randomly mutating and screening the key enzymes in these metabolic pathways, we could increase the production of the anti-aging compound spermidine and the antioxidant NADPH, and also can let it degrade more nicotine in E. coli. First, we need to combine the second-generation black box with the three plasmids we constructed that contain the key enzymes and transform them into BL21 for mutation. Through sequencing analysis, we can determine whether our second-generation black box system can function properly in these environments.
(2)Build
We constructed plasmids for spermidine synthesis (spermidine synthase, S-adenosylmethionine decarboxylase, and agmatinase) and NADPH synthesis (6-phosphogluconolactonase, transaldolase, and transketolase), and co-transformed them with the second-generation black box plasmid into E. coli BL21. For each experiment, we established a control group (where the addition of water would not activate black box) and an experimental group (where the black box was activated by IPTG and exposed to blue light). After the second-generation black box mutated the synthesis plasmids, we performed sequencing analysis on the control and experimental groups.
(3)Test & Learn
Figure3. Testing results for Spermidine and NADPH synthesis Pathway
Figure4. Testing results for Nicotinate Degradation Pathway
The results revealed that, compared to the control group, the experimental group exhibited a clear C-to-T substitution, indicating the successful mutation of the three plasmids by the second-generation black box, with the majority being C-to-T mutations.
5. Cycle 5 Safety Consideration before Application on Advanced Life
(1)Design
To address the potential issue of gene leakage from host cells, we developed a suicide system to trigger cell death if the cells leak into the environment. We encoded an enhancer-containing plasmid that encodes essential enzymes for ATP synthesis. This plasmid is regulated by an arabinose repressor protein produced from an upstream sequence, which binds to the plasmid and prevents transcription of the downstream gene. If the cells leak into an environment containing arabinose, the arabinose will bind to the repressor protein, initiating the transcription process. Once RNA polymerase (RNAP) reaches the coding sequence, it will cleave the sequence responsible for producing the essential ATP-synthesizing enzymes, effectively disabling the cell’s energy production.
(2)Build
Figure4. Regulatory sequence for safety (With Arabinose)
Figure5. Regulatory sequence for safety (Without Arabinose)
There are concerns about the potential leakage of genetically modified host cells into the environment. To address these, we designed a suicide system that triggers cell death upon environmental exposure. The system includes a plasmid enhancer that codes for essential enzymes involved in ATP synthesis. An arabinose repressor protein, produced from an upstream sequence, binds to the operator, preventing transcription. A Cas9 gene is placed downstream of the promoter to halt protein production if transcription is triggered. When the host cell is leaked into an environment containing arabinose, the arabinose binds to the repressor protein, initiating transcription. As RNA polymerase reaches the Cas9 sequence, the Cas9 protein is expressed and cuts the ATP-synthesis enzyme-coding sequence, effectively shutting down the cell's energy production and killing it. This positive control system ensures safety by preventing the survival of genetically modified cells outside their intended environment.
(3)Test & Learn
As a result of our built-in safety mechanism, we did not see the growth of E. coli outside of the laboratory.
6. Cycle 6-Implementation of Caenorhabditis elegans
(1)Design
Since we cannot directly experiment on humans, we chose an alternative organic organism for testing—a well-suited nematode. Given that spermidine has anti-aging effects, NADPH has antioxidant properties, and nicotine is toxic, all of these factors may directly or indirectly affect the lifespan of the nematode. Therefore, we can design a series of related experiments by feeding the nematodes with those winner mutants we have selected in the previous experiments to verify our hypothesis by observing and statistically analyzing the average lifespan of the nematodes.
(2)Build
For the spermidine synthesis experiment, we set up a control group (fed nematodes with normal BL21) and an experimental group (fed nematodes with well-mutated BL21). We placed the nematodes on two LB plates containing the same amount of arginine. Since we predict that the nematodes fed with mutated BL21 will produce more spermidine, leading to a longer lifespan, the final data for the average lifespan of nematodes from the experimental group should be higher than that of the control group.
Similar to what we did in the previous experiment, for the NADPH synthesis experiment, we set up a control group (fed nematodes with normal BL21) and an experimental group (fed nematodes with well-mutated BL21). We placed the nematodes on two LB plates, both containing the same amount of H2O2. Since we predict that the nematodes fed with mutated BL21 will produce more antioxidants, and the extended lifespan of the nematodes would confirm this, the average lifespan of the experimental group should be higher than that of the control group.
In the nicotine degradation experiment, we also set up a control group (fed nematodes with normal BL21) and an experimental group (fed nematodes with well-mutated BL21). We placed the nematodes on two LB plates, both containing the same amount of nicotinate solution. Since we predict that the nematodes fed with mutated BL21 will degradate more nicotine, the average lifespan of the experimental group should be higher than that of the control group.
(3)Test & Learn
We tested their lifespan and found that the nematodes that consumed the mutated bacteria had a significantly longer lifespan. This suggests that the mutations in the bacteria positively impacted the health and longevity of the nematodes.
Figure6. C. Elegans Observation
Figure7. A）average lifespan of nematodes in spermidine and NADPH synthesis experiments B）average lifespan of nematodes in nicotine degradation experiment
7. Cycle 7-Construction of a Mutation Library Database Design
By leveraging this technology, we are able to do a semi-random mutation on genes from animals and humans. The next question is how to select those genes with "gain of function" mutation?
Here, since the fluorescence of GFP may change when mutagenesis is conducted on key genes, we utilized it as a real-time reporter and developed a high-throughput screening method based on fluorescence, employing fluorescence-activated cell sorting (FACS) and Next Generation Sequencing. However, while this process can help track the progress of mutagenesis, it doesn't provide information on the actual quality of the mutations.
For other metabolic genes we want to optimize, we use different selective conditions in LB plates to identify "gain-of-function" mutations. Colonies that are GFP-negative but survive selection are processed using our fluorescent-based high-throughput screening method, and specific sequencing results are identified as "gain-of-function" mutations. We further confirmed the functional mutations by feeding Escherichia coli with mutated proteins to nematodes who grew in environments intended to benefit nematodes with the desired “gain of function mutations.”
These beneficial mutations can be recorded in our Dictionary System. As we continue to accumulate knowledge on mutation-function relationships, this system will become increasingly useful for guiding gene therapy in the future!!
Figure8. Project Overview
8. Cycle 8-Potential Future Application in the medical field
(1)Design
The use of probiotics has shown positive effects in improving the health of C. elegans (nematodes), leading to the question: could this be applied to humans as well? If the benefits observed in these organisms can be replicated in humans, it opens up the possibility of developing a product that specifically targets human health. We could design a probiotic-based solution tailored to enhance well-being by focusing on mechanisms that promote healthy gut flora or other beneficial biological processes in humans, building on the promising results seen in model organisms like nematodes.
(2)Build
We introduce this mutated plasmid into Lactobacillus to create a Lactobacillus probiotic urachus.
Figure9. Potential Applications of Black Box System
(3)Test & Learn
While we have not yet tested this capsule, it has already been synthesised in collaboration with our partner company. This capsule is designed to enable the gut microbiota to produce more spermidine. We expect to see an effect in humans, but further experiments are needed to confirm the results. Stay tuned for updates!