PROOF OF CONCEPT
Our iGEM project consisted mainly on the development of the necessary DNA parts for the generation of genetic circuits within the Chlamydomonas reinhardtii chloroplast. A chassis novel to the iGEM competition but which harnesses a massive potential for the production of recombinant proteins or interference with the multiple metabolic pathways which occur in the organelle.
We aim to provide a comprehensive library of parts which allows future users to conduct exciting projects. These parts adopt the new phytobrick standard introduced into the competition this year. The proof of concept of functionality of our parts as a new library was shown by the assembly of higher level composite parts and then confirmation of correct assembly through restriction digest.
This, in principle, straightforward task had many challenges associated. Firstly, the genetic system of Chlamydomonas chloroplasts is highly A/T biased and thus parts were challenging to synthesize with iDT technology. Additionally, the inclusion of the appropriate overhangs (misguided by the documentation) lead to the need to PCR amplify each fragment to include BsaI restriction sites, which lead to a tremendous effort of PCR troubleshooting to manage primers which anneal to such an A/T rich sequence. Further, our parts library would not be practical without the inclusion of homology regions to flank the cassette with the gene of interest as constructs are inserted into the genome in plastids through homologous recombination. After consulting the expert Ralph Bock from the Max Planck Institute of Molecular Plant Biology and our advisors here at the department of Plant Sciences of the University of Cambridge we were confirmed that the size of the homologous regions needed to be at least 1 kb long. This posed the challenge of PCR cloning and thus was yielded by assembly through golden gate.
In conclusion, we have proved our new Chlamydomonas chloroplast parts library which is functional and amplifiable in competent E. coli cells. Please read about other concepts proved in our project in the hardware and modelling development as well!
We have managed to create a low-cost, DIY, gene gun that would fire DNA into plant cells. The complete assembly instructions, materials required, and an example of it working under real world condition can be found here
We managed to write a code that would enable anyone to predict the time taken to achieve homoplasmy using the appropriate model for Cas9 activity. This program is also designed to be flexible in the sense that various input parameters can be changed to fit the parameters of your experiment. For more information, please read a complete documentation of the program under the 'Modeling' page.