Team:UCAS-China/Future Plan

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Future plans

Temperature-controlled Switch Testing

The temperature-sensibility switch is a vital part of our design and accounts for the biosafety function. The switch is based on the project conducted by the team of UCAS in 2019, which has been well tested, and we made a significant innovation by connecting the Doc gene, which instructs the translation of toxin protein, with intracellular degradation tag SsrA. The upgraded switch is predicted to achieve a goal of extremely low escape rate, meanwhile, to avoid accidental suicide or plasmid loss during the reproduction of our engineering bacteria.

Our future work would ideally prove the predictions mentioned above by transforming the plasmids containing the temperature-sensibility switch into L. acidophilus. Then the bacteria will be cultivated in the environment of 37℃until the quorum density is high enough. Afterwards, the bacteria liquid will be divided into two parts, one of them is used to extract protein through western blot method to test the content of protein Doc; the other part is transmitted into environment of 25℃ which represents room temperature and kept been cultivated, then the density of L. acidophilus will be tested. We can also evaluate the activity of DOC toxin protein and the effect of gene circuit by detecting the solid escape rate. By culturing L. acidophilus at different temperatures, we can test the effect of temperature sensitive switch.

Validation of The Entire Design

In essence, it must be ascertained that, when cultivated at 37℃ and the ammonia concentration reaches a certain threshold, our chassis can release suitable antimicrobial peptide in the extracellular space to limit the growth of H. pylori while avoiding completely removing it. Finally, the function of the temperature-controlled switch should be guaranteed.

In order to achieve this goal, we introduced ammonia concentration sensing switch and antimicrobial peptide secretion circuit into two different plasmids. Then, it is necessary for us to do the transformation procedure twice to equip our chassis with two kinds of plasmids mentioned above. We plan to firstly validate the entire design using E. coli because we have rich information about its culture. If the preliminary experiment is successful, we will use microfluidic technology to simulate the acidic environment of the stomach to culture and further detect L. acidophilus. We believe our chassis will be fully competent for our goal at that time.

Co-culture of L. acidophilus and H. pylori: Medium Environment & Gastric Environment

In order to validate the therapeutic effect, the co-culture experiment should not be skipped.

In the first stage, we will use microfluidic technology to construct an acidic environment simulating human stomach before we put H. pylori and our chassis in succession into it in a certain proportion (the proportion will be determined considering the distribution of stomach flora and the result of our modelling). We hope to see a result that, when our chassis are put into the medium after H. pylori has a considerable number, the density of H. pylori experience a sharp decrease, ultimately the two strains achieve dynamic balance in a long run with the density of H. pylori been controlled at a low level.

As we all know, it is impossible to simulate the environment of the stomach exactly the same. Here comes the need for animal experiment. With the approval of the school and the iGEM project committee, we plan to do the co-culture procedure in the stomach of living mice. We will stain the two strains with different dyes and inoculate them into the stomach of mice. Through regular gastric juice sampling and analysis, we can obtain the specific data of the population changes of the two strains in the stomach of mice, and compare them with our simulation results.