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Professor of Chemical Engineering and engineering biology University of Texas at Austin
Prof. Hal Alper developed novel synthetic biology and directed evolution approaches to increase the capacity of engineered cells. He has developed a similar in vivo continuous evolution (ICE) system in yeast using Ty1 transposon and an encoded reverse transcriptase. After we decided on our project, we contacted him through mail regarding problems on our experimental design. He affirmed to us the necessity of adding capsid protein to the system as it serves to increase reverse transcription efficiency. He also told us that poly-purine tract is needed for reverse transcription to take place, so we added the relevant sequences to our design.
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Professor of microbiology and microbial engineering Fudan University
Dr. Zhong provided us with advice on improving our design by refining the details. In our project we designed a series of experiments to verify our system and insure its feasibility and efficiency. He pointed out to us that apart from the system’s overall function, we also need to set up experiments for the testification of each process in our system. It’s the accuracy and accomplishment of each step that ensured our system’s function.
Dr. Zhong also reminded us that the expression of reverse transcriptase (RT) might be affected in a heterologous host, and that the high expression of RT may cause misfolding and the formation of inclusion body. This led us to test which promoter is best suited for the expression of RT.
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iGEM Asia Ambassador. Member of iGEM Human Practice Committee
Our meeting with Ms. Zhang corrected one of our biggest misunderstandings with iGEM competition and the presentation of our project. Our initial belief was that the project could be separated into three different parts—experiment, modelling and human practice. We thought of carrying them out separately and then mix the results together at the end. She pointed out that this is totally incorrect, and emphasized to us that the ‘three’ parts are actually embedded with each other. They both offer each other guidance and refine themselves in this process. With this in mind, we chose to interact with our project’s end users, and refine it upon the receival of various suggestions.
In addition, she also reminded us that we’re doing human practice for a reason,every activity needs to have its meaning, that we’re not acting in order to fulfill the medal criteria, but to make our project better. We have designed our human practice work based on this principle.
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Professor of genetics with a focus on molecular genetics and genetic engineering of yeast Fudan University
At the beginning of our project design, we were debating over which means should be used for system verification. One was to measure the expression of fluorescent protein, which should be recovered by the correct point mutation; the other was by utilizing antibiotic genes, successful recovery could be demonstrated by the growth of colonies. Both constructs have its advantages and disadvantages. During the interview with Dr. Lv, she pointed out that fluorescent detection would be less accurate and more laborious than colony growth, which is also easier to detect. She also mentioned that we could simulate the evolution of resistance genes or construct E. coli strains with attenuated or disabled endotoxin as future application.
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Researcher of genetics with a focus on genome editing technique Fudan University
We encountered problems when co-transforming the plasmids carrying Cre and loxP-flanked mCherry respectively. We found that the fluorescent plasmid was undetectable even after PCR amplification. After our talk with our PI, we supposed it is the leakage of Cre that leads to the problem. Dr. Wang offered us various suggestions on how to enable a more stringent control of Cre expression, including using less strong promoter and overexpression of regulatory protein.
Initially, we were using same loxP sites not only in Cre activity tests, but also in our system design. Dr. Wang warned us that the splicing rate of Cre is much higher than its recombination rate. If we continue to use the same loxP sites on both ends, the DNA fragments are likely to self-splice instead of initiating recombination as we hoped. This is affirmed by Dr. Alper, who also mentioned that the multiple plasmids containing the loxP sites in the system could interact with each other. Two ideas were offered by Dr. Wang to solve this problem. One is using the mutated loxP site on one end of the DNA fragments and another is to use different recombinases such as Cre and Flp together. Adopting his suggestions, we changed one of the loxP sites into its mutated incompatible versions and tested their feasibility.
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Professor of genetics Fudan University
Prof. Lin mentioned to us that even if we controlled the expression of Cre, we could not be certain of the length of time it will stay in the cell. So, after we removed the inducer from the culture, Cre would likely continue to function, thus resulting in a mismatch between survived cell phenotype and unrelated genotype. Cre could recombine our desired sequence to the plasmid for one time and allow the cell to survive our selection, but recombine again afterwards and replace it with other versions when we’re scanning the plasmid. He suggested that we make efforts to address this problem, and this is how we came up with the addition of degradation tags to Cre.
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Professor of genetic engineering focusing on structural biochemistry of gene editing systems and frontier technologies Fudan University
When we were unable to model the whole reaction process of the reactions of R-Evolution, we contacted Prof. Qiang Huang for suggestions. He suggested us modularize the reactions and model the reaction process one by one, using the output of the previous model as the input of the next one. He thought it a reasonable way to approximate the real reaction, where different reactions mingle together. He also suggested that we could do some Monte Carlo simulation to acquire more information, such as the noise of the reactions. We readily adopted his advice and came up with the present models, from which we surprisedly found that the expression of Cre and reverse transcriptase should be differentiated which helped us a lot with our experiments.
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Researcher of genetics, expert in Adeno-associated virus (AAV) Fudan University
As an expert in Adeno-associated virus (AAV), Ling showed great interest in our project and thought it promising. However, he also expressed his queries and suggestions. His main concern was the proportion of the native plasmids within the system after mutagenesis. In his opinion, it is an important indicator to evaluate the efficiency of our system. We tested this problem through modeling and found the optimal environment for the highest recombination rate to occur.
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Professor of Chemical Engineering and engineering biology University of Texas at Austin
Upon our invitation, he visited our university in August and held a workshop with us. In the workshop, He gave us a lot of valuable advice as both a researcher and user of directed evolution. To start with, he pointed out that besides cheaper and less labor intensive, another big advantage of our system is that it can be adapted across various hosts. He also affirmed other problems that were mentioned before, including the leakage of Cre and the self-splicing of our mutated DNA fragments when using the same loxP. Besides, he held a lecture on synthetic biology in our school which was attended by many students including both undergraduates and graduates.
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