Implementation

The motivation for the development of random mutagenesis is due mainly for the intention of solving problems in a macro level. Random mutagenesis could either enhance or undermine the expression of genetically coded products, so by implementation of random mutagenesis, selection of the winner mutation, and finall product developement we can use the most effective mutation it in many aspects, in turn solving many issues.

1. Semi-random mutagenesis first generation VS Controllable random mutagenesis second generation

Figure1. The Second generation of controllable mutagenesis system

Using this intuition as a template, we develop a crude system consists of Lac operon, CBE (cytidine deaminase) and a functional T7 RNAP (RNA polymerase). This system could mutate cytosine to thymine from the T7 promoter to the terminator, but due to its uncontrollable nature, the CBE mutating all of the cytosine in its path to thymine, the first generation system couldn't be satisfactory for a universal problem solving method.

Due to the ineffectiveness of the first generation mutagenesis system, we aim to induce more control over the whole process of random mutagenesis. By exploiting previous experiences, we introduce light sensitive components including N-terminal RNAP, C-terminal RNAP, n-Mag, and p-Mag to the current T7 RNAP. In the new light sensitive T7 RNAP, blue light could make the components of the RNA to bind together to form a functional RNAP, making the transcription process proceed. By utilizing control over the time of transcription, we can effectively select the most successful gene in promoting a specific process.

Figure2. High throughput screen for selection in florescent intensity

2. Human selection VS High throughput screening selection

The construction of the system is then encoded in to plasmid for transformation. Human selection selects the most successful host cell by using florescence microscope for observation and choosing the most prominent mono clone colony. However, this technique is inefficient for macro scale selection. To overcome this effect, we turn to the use of high throughput screening. High throughput screening provided a pathway to effectively select large amount of cells. In this case, we could first put the cell in an environment that is harsh and very selective to enhance plasmid production. for example, genetically modified Estrecholia coli (E. coli) could express high amount of plasmid growth in a petri dish that contained over twenty percent glycerol and has a ampicillin selection. This host cell will then undergo the second generation system and put in to cytometry machine for the selection of the cell that has the lowest florescence 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. Moreover, performance screening is achieved as cells are selected after they are also put in environments favoring desired "gain of function" mutations.

Figure3. Spermidine metabolic pathway

3. Plasmid construction

Different plasmids code for various enzymes in a metabolic system. Demonstrating a project 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 in our project are for this use:

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 that could be responsive for random mutagenesis.

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 alternating essential enzymes by our project, this will demonstrate of the viability of our implementation to the cell. Therefore, we constructed this pathway.

Overall, construction of plasmid for implementation of the system could be an essential way for our project’s effectiveness as it could provided substantial evidence for our system is practically implemented.

4. safety

Figure4. Regulatory sequence for safety

There are some concerns about the leakage of genetically modified host cells in to the environment. To address these concerns, we developed a suicide system for the cell after it is leaked in to the environment. First, we encode an enhancer containing plasmid that coded for essential enzymes for ATP synthesis. The operator will be bind by the arabinose repressor protein produced in the upstream sequence, and there will be cas 9 gene directly after the promoter to prevent the protein after transcription process is initiated. When there is a leak, the arabinose outside the original environment will bind to the arabinose repressor protein to initiate the transcription process. Then after RNAP reaches the cas 9 sequence, the cas 9 sequence will cut the original enzyme coded sequence, “blowing up” the energy factory for the host cell. This is a positive control system of which we implement our safety issues on the cell.

5. Could meet up to expectations?

Figure5. Practical application of spermidine metabolic pathway in Caenorhabditis elegans

To test the validity of our project, we implemented the system on a previous stated metabolic pathways to provide material evidence. With implementation of another pathway of transforming spermidine to arginine, we could discover the effect of our system on more complex organism, in preparing for other organisms. With similar process above, the key mutation for the enhancement of arginine production is registered as a substantial template for future use. To visualize the effect of this mutation, we provide the genetically engineered E. coli for C. elegans consumption, as there is a known positive connection between arginine and C. elegans lifespan. As predicted, the life span of C. elegans is elongated substantially to the control group. Overall, we testify the use of our system substantially by discovering the most successful mutations and visualizing it in a practical context. In addition, enzymes in the NADPH synthesis and nicotinate degradation pathways are very much optimized as well.

6. Documentation of winner mutation

Figure6. The winner mutation of GFP after comparing sequences

By sequencing the selected host cells, we can compare and contrast the original sequence and the mutate sequence. Then by using alphafold 3 to predict protein structure, we can see the specific alteration of enhancement in the protein. The example of a change in conformation that results in the enhancement in function is the GFP mutation above. By documenting it, we can create a template for others to utilize for their project. This that function as a dictionary could contribute to our project’s practical implementation in helping the public and Repeated action could be taken for the further benefit of others in a macro level.

7. Implementation on end product

With the implementation of our system on the end products in the last chain of industry, we visited a pharmaceutical company for cooperation. We introduced the system in their Probiotics by enhancing the production of products inside the host cell, resulting in a better function. This cooperation is still in negotiation, but we believe that a bright future of collaboration between firms and researchers is waiting to be implemented. We believe in practical implementation and we will implement our design practically.