Team:FAFU-CHINA/Results

This year, FAFU-CHINA team focused to utilize CC530 Chlamydomonas reintmrdtii to express Cry and Cyt genes which cloned from BRC-LLP29 Bacillus thuringiensis strain to kill the larvae of mosquitoes. This project owns various advantages expect for the express platform based on Chlamydomonas reintmrdtii. We also improve the toxicity of Cry and Cyt and improve parts as following.1. Firstly, we decide to express Cry4a，Cry10a，Cry11a，Cyt1，Cyt2 genes which cloned from BRC-LLP29 Bacillus thuringiensis strain in E. coli. Due to the homologies of Cry genes, we were stocked in cloning. With the support from ShanghaiTechChina_B team, we constructed Cry11a-pET32a (+) and Cyt1-pET32a (+) express vectors. And tested the expression of Cyt1 by SDS-Page.2. Firstly, we decide to express Cry4a，Cry10a，Cry11a，Cyt1，Cyt2 genes which cloned from BRC-LLP29 Bacillus thuringiensis strain in E. coli. 3. Synthesizing Cry and Cyt toxin genes based on Chlamydomonas reintmrdtii codon-optimized to improve the express level.4. To increase toxicity to the larvae of mosquitoes and the potential possibility of resistance, we utilized 2A peptide system to co-express Cry and Cyt toxins.5. Based on the different toxicities of Cry and Cyt, we contrasted six toxin groups to select the best group.6. The efficiency of Hsp70A-Rbc S2 promoter can be improved when environmental temperature is larger than 40℃. It means that the toxins express device will express more toxins in summer. Meanwhile, with the help from NEU-China, we tested the heat shock induced result by q-PCR and confocal laser scanning microscope (CLSM).7. Based on the bioinformatics analysis, the result showed that the GC% was significantly different between Bacillus thuringiensis and Chlamydomonas reintmrdtii. So with the technology support by Genes for life-DNA Company in Suzhou, we optimized EGFP，Cry4a，Cry10a，Cry11a，Cyt1，Cyt2 genes based on codon optimization. We standardized these genes into psb1c3 parts and submitted them. You can find more information by this link:8. Based on the express vector pChlamy-3 which provide by Huiying Zhang, we constructed 13 express vector in Chlamydomonas reintmrdtii.9. Bacillus thuringiensis is hot spot in previous iGEM competition. There are various Cry genes which reserved in Parts stock. Based on the toxin express platform in our project, we improve NCTU_Formosa BBa_K332011 parts which encodes Cry11Aa by codon optimization. And we submitted the improved part BBa_K2074023.Meanwhile, we also improved BBa_E0040 part which encodes wild type GFP. And we submitted BBa_K2074026 which encodes eGFP. These improvements can help another teams to utilize Chlamydomonas reintmrdtii. We also helped ShanghaiTechChina_B, BNU-China and BNU-China team do experiments and offered a protocol to JFNLS_China. You can find the details in the experiments wiki pages.