This year, FAFU-CHINA team focused toutilize CC530 Chlamydomonas reintmrdtiito express Cry and Cyt genes which cloned from BRC-LLP29 Bacillus thuringiensis strainto kill the larvae of mosquitoes.
This project owns various advantagesexpect for the express platform based on Chlamydomonasreintmrdtii. We also improve the toxicity of Cry and Cyt and improveparts as following.
1. Firstly, we decide toexpress Cry4a，Cry10a，Cry11a，Cyt1，Cyt2genes which cloned from BRC- LLP29Bacillus thuringiensis strain in E.coli. Due to the homologies of Crygenes, we were stocked in cloning. With the support from ShanghaiTechChina_Bteam, we constructed Cry11a-pET32a (+) and Cyt1-pET32a (+) express vectors. Andtested the expression of Cyt1 bySDS-Page.
2. Firstly, we decide toexpress Cry4a，Cry10a，Cry11a，Cyt1，Cyt2genes which cloned from BRC- LLP29Bacillus thuringiensis strain in E.coli.
3. SynthesizingCry and Cyt toxin genes based on Chlamydomonasreintmrdtii codon-optimized to improve the express level.
4. Toincrease toxicity to the larvae of mosquitoes and the potential possibility ofresistance, we utilized 2A peptide system to co-express Cry and Cyt toxins.
5. Basedon the different toxicities of Cryand Cyt, we contrasted six toxingroups to select the best group.
6. Theefficiency of Hsp70A-Rbc S2 promoter can be improved when environmentaltemperature 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 inducedresult by q-PCR and confocal laser scanning microscope (CLSM).
7. Basedon the bioinformatics analysis, the result showed that the GC% wassignificantly different between Bacillusthuringiensis and Chlamydomonasreintmrdtii. So with the technology support by Genes for life-DNA Companyin Suzhou, we optimized EGFP，Cry4a，Cry10a，Cry11a，Cyt1，Cyt2 genes based on codonoptimization. We standardized these genes into psb1c3 parts and submitted them.You can find more information by this link:
8. Basedon the express vector pChlamy-3 which provide by Huiying Zhang, we constructed13 express vector in Chlamydomonasreintmrdtii.
9. Bacillus thuringiensis ishot spot in previous iGEM competition. There are various Cry genes which reserved in Parts stock. Based on the toxin expressplatform in our project, we improve NCTU_Formosa BBa_K332011 parts whichencodes Cry11Aa by codonoptimization. And we submitted the improved part BBa_K2074023.Meanwhile, wealso improved BBa_E0040 part which encodes wild type GFP. And we submittedBBa_K2074026 which encodes eGFP. These improvements can help another teams toutilize 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.