Team:XHD-Wuhan-Pro-China/Contribution
Contribution
Literature Review
Through reviewing literature, we have added information to the Registry page for six existing parts: BBa_I712667, , BBa_K3512002, BBa_K857000, BBa_K1602049, and BBa_K3187028
1. BBa_I712667: HIV-1 aspartyl protease- We found studies on the mechanism of HIV-1 protease and the expression of HIV-1 protease in E.coli. We hope that future teams who would like to develop HIV inhibitors or have the need to express the HIV-1 protease gene in E.coli will find our contribution useful.
2. BBa_K3512001 and BBa_K3512002: CcdB Toxin and CcdA Antitoxin- These two parts are usually used together. They form the CcdA-CcdB toxin-antitoxin system. We found a study on variants of CcdB and CCdA in E.coli, the experiment of reconstituting the inactive GyrA-CcdB complex, and the research on how the conformations within CcdA’s free-energy surface enable it to both bind CcdB and regulate cleavage by Lon protease. We hope that our contributions will help future teams construct better kill switch, using the CcdA-CcdB toxin-antitoxin system.
3. BBa_K857000: acetaldehyde dehydrogenase- This is a gene that we use in our project. We found studies on whether the heterologous ATP-independent A-ALD and PFL pathways can support the growth of acs1 acs2 mutants of S. cerevisiae by providing extramitochondrial acetyl-CoA. We hope that our research will help future teams who also deals with the alcohol related issues better construct their circuits.
4. BBa_K1602049: Rrkey- For this part, we found studies that focus on regulatory circuits operating within 5′ UTRs and specifically discuss the role of ribosome binding signals, translational repressors, low molecular weight effectors, noncoding RNAs (ncRNAs) and temperature as parameters affecting mRNA stability. We hope, with this new information, future teams will find it easier to use this riboregulator-system in their project design.
5. BBa_K3187028: Sortase A7M- For this part, we provided a thorough introduction about the functions of sortases in bacteria. We also mentioned site-specific protein labeling via sortase-mediated transpeptidation. We hope that by reading our review, the biochemical process regarding sortases will become clearer to the future teams, so they will make better use of it.
Acceleration Module
In our project design, we make of the nadE and nox gene to form the acceleration module. The nadE gene will be expressed as NAD synthetase which amidates the Nicotinic adenine nucleotide (NaAD) into NAD(H), maintaining the ratio of NAD+ to NADH at an appropriate level. Meanwhile, the nox gene will be expressed as NADH oxidase, promoting the oxidation of NADH into NADH+ and restoring the balance between NAD+ and NADH. The system will not only help accelerate the reaction of alcohol and acetaldehyde dehydrogenase but also speed up many other reactions of dehydrogenation as well as oxidation. We hope that the acceleration system that we construct will keep benefiting the future teams, helping them to more efficiently achieve their goals.
The use of E.coli Nissle 1917
Following the development of synthetic biology and further study about intestinal microbiota, more and more scientists want to construct genetically engineered probiotics and make them function in the intestines. However, little is known about the genetic background of the common probiotics, which poses great challenges to the process of genetic modification. Besides, ordinary E.coli strains contain endotoxin, making them unsuitable for carrying out their functions in the intestines of human or animals. Therefore, we choose E.coli Nissle 1917 since the majority of its genetic information has been already available to us, and it does not any endotoxin.
In our project, we tried using E.coli Nissle 1917 as our chassis creature, and we have successfully constructed a genetically engineered strain of this bacteria that could normally carry out all the expected functions. We hope that our successful engineering of E.coli Nissle 1917 will help convince future teams to use this bacteria as well and provide them with valuable experience regarding its usage.