Human Practices

what we gain

1. Potential benefits of GenOMe compares to other gene editing methods
2. Obstacles GenOMe could met when apply in research or factory
3. What benefits we should focus on when promoting our topic

record

The professor introduced existing gene editing technologies and generously explained the various technological breakthroughs of the past decade. After absorbing his stories, he offered several suggestions based on our bottlenecks:

1. Although shifting gene editing methods to the genome won't completely resolve instability, when an organism's plastids have been edited to their limits, genome editing can add functionality to the edited organism.
2. Focusing on the technical reasons for not editing the plastids and avoiding antibiotic selection is feasible, and this technology can also provide a convenient alternative for subsequent teams.
3. Build a Demonstrate Library. Suppose you want to design multiple genes for simultaneous expression but are unsure how to modify each gene to achieve optimal expression. In this case, you can create a library and simultaneously introduce gene fragments of varying expression levels into the genome. Then, use different promoters to express different genes with varying strengths, saving the time of individually editing each gene. This concept can be verified by simultaneously editing GFPs of varying intensities and using different promoters to achieve simultaneous expression of varying brightness. Rather than being concerned about the inability to delete genes, our technology should focus on large-scale gene editing. Furthermore, our technology offers many advantages for industrial strains, such as reducing the need for antibiotic administration and plasmid loss.
4. Regarding PCR primer design, we have a discussion of how it can be examined on a larger scale. Our hypothesis at first is that since there is research about ORBIT or extracellular two-to-two experiment, which was integrated by PCR fragment, we assume PCR fragment two-to-two experiment can work intracellularly. However, professor Paul has mentioned that PCR fragment can’t be used intracellularly due to rapid degradation. To solve this problem, he mentioned that if the integration efficiency of BxB1 is high enough, we could successfully integrate intracellularly and significantly improve the efficiency of modification overall.

what we gain

1. What are our benefits and shortcomings compared to CRISPR or other gene modifying methods?
2. What is the value of building a Demonstrate Library?
3. What potential applications can our topic have?

record

We have contact with the iGEM team NCKU team, and after we have our discussion about the obstacles and solution we both met, the team leader of NCKU mentioned that their professor, I-Son Ng, is interested in gene modifying. Hence, we followed his recommendation and have an interview with professor, the following are recommendations during the interview:

1. For issues of insufficient copy number and low fluorescence intensity in PCR verification experiments, these can be addressed by using promoter 7 or increasing the number of GFP genes to enhance fluorescence.
2. This technology can be used to disrupt specific gene pathways; for example, to restore functionality in bacteria that produce acetic acid, a gene can be inserted into the acetic acid-related pathway. This allows our technology to simultaneously perform both blocking and production functions, improving experimental efficiency.
3. While the concept of reducing cellular burden is feasible, avoiding the use of antibiotics remains extremely difficult. Using antibiotics in conjunction with GFP can increase the accuracy of screening.
4. The idea of ​​a "Demonstrate Library," which allows simultaneous expression of different genes, is appealing, but regardless of the results, individual sequencing is still required, making it difficult to utilize. Focusing on different insertion sites can create a library of various strains, which can aid in studying metabolic profiles.
5. Since our research organism is E. coli, we can focus on the acetic acid and lactic acid it produces during growth. Our technology can therefore be used to avoid interfering with its metabolic processes.

what we gain

1. Our model originally used concentration as its input, but since it's difficult for wet lab experiments to provide accurate data on intracellular concentrations, we are looking for alternative methods to validate our model.
2. Can we enhance the accuracy of our model by incorporating AI?
3. How can we combine experimental data with the model's predictions?

record

To find experts who can help us solve problems in dry models, we ask for help from professor Tzong-Yi Lee who is an expert in bioinformatics. The professor had helped our former and former teams, so he is not a stranger to the Biobrick or dry model, however, he is not familiar with the model we required since our experiment is abnormal compared to former teams. No matter how, he still give us some advice from his experience:

1. A prerequisite for building a model is, of course, having sufficient experimental data. We can start by investigating sequence data and the related protein structures of DNA to understand the model's architecture.
2. Our model doesn't require AI to analyze data; instead, it uses simple mathematical formulas to calculate the required data based on binding concentration and time parameters.
3. Initially, we hoped to adjust the experimental data to match the predicted values. However, the true value of the model lies in its ability to adjust the predicted values ​​after analyzing the data to better reflect the actual results. The workflow between "wet" and "dry" experiments should be that the "dry" model first provides a prediction, and then“dry" uses the obtained data from the“wet”experiment to regulate the coefficients in the prediction formula. Therefore, there's no need to worry if the experimental data doesn't perfectly match the prediction.
4. For our "two-to-two" model, its formula is certainly different from that of a "one-to-one" model, but there is likely a relationship between them. Since the "two-to-two" model has two binding sites with different affinities, it will have two different protein concentration coefficients. However, because of these differences, we can compare them to determine which has a greater impact. By deriving the relationship between the two, we might obtain a more accurate equation to explain the dual-site binding. Furthermore, we can explore whether there is a third variable in the formula, besides the two concentration variables, and whether there are interactions between them.

what we gain

1. value of GenOMe in the perspective of researcher
2. the best benefit of GenOMe
3. applications of GenOMe in experiment or extreme environments

record

Through the team leader's introduction, we met Dr. Jiang Yinling, who offered the following advice during our visit:

1. Using ORBIT simply for insertion is of limited use.
2. If some essential genes are required, we can insert them after them. This eliminates the need for a promoter or other mechanism, as that region will automatically activate anyway. This deactivation mechanism prevents over-repressing and improves switching performance.
3. When selecting a harbor, choose one above the resistant gene. This way, antibiotics are not required and only applied at the end of the insertion, resulting in a cleaner design. This is also convenient for future pharmaceutical strains, as the clean design prevents the development of drug resistance and makes it easier to control.
4. The PCR concept is feasible and can be developed in a similar manner to long-distance PCR. After PCR, separate genes can be ligated together, allowing for simultaneous PCR and BXB1 insertion.
5. The convenience of GenOMe has been confirmed, as well as its applicability to non-model bacteria such as mammalian cells and nematodes. While CRISPR is still relatively inexpensive, it's not suitable for long gene fragments, and GenOMe also retains directional selectivity and irreversibility.
6. Projects where our topic is applicable include the following:
   1. To enhance drug resistance, large numbers of identical resistance genes are co-edited and placed into chromosomes, significantly increasing bacterial resistance.
   2. Studying bacterial conditions without interference from plasmids.
   3. Clinical strains, such as next-generation probiotics for pharmaceutical production.
   4. Under extreme environments (e.g., energy requirements), minimizing plasmid editing to increase stability and reduce cellular burden.
   5. Increase the advantages of the bacteria themselves, such as reducing the carbon emissions of negative carbon organisms themselves.
7. The focus can be on the benefits of chromosome editing, such as our ability to stably integrate large gene fragments into chromosomes after the plasmid has been edited to increase their value.