Team:Cambridge-JIC/Results
RESULTS
It has been a summer of a lot of work, many mistakes and many corrections. Below you will find a bullet points of the broad overview of the successes and less successful aspects of our wetlab project. We then elaborate in text our experience and where we put our effort in. We have included “failures” and “mistakes” as we consider these to be useful to future iGEM teams who brave the challenge of working with plastids!
What we achieved this summer
- Generated a library of parts for Chlamydomonas reinhardtii chloroplasts
- Verified these parts through sequencing and/or restriction digest
- Verified our aadA antibiotic cassette in E.coli
- Biolistically transformed Chlamydomonas with gene gun
- Library-based design of homoplasmy strategy for chloroplasts
Outside the wetlab our work this summer has lead to
- DIY biolistics gene gun
- DIY Growth facility
- Modelling software for experimental design
What we did not achieve this summer
- Achieve homoplasmy in chloroplasts with our genes of interest
- Were not able to establish a positive strain expressing a fluorescent protein
- Did not express cas9 in chloroplasts to study their toxicity in this chassis
- Did not test our homoplasmy strategy in vivo
Future plans for the project
- With available parts we provide, establish positive strains for more complex experiments
- Express cas9 linked to VFP-HA to study toxicity
- Apply and debug the homoplasmy acceleration strategy
- Optimization of protocols for transformation with our DIY gene gun
Excited about the potential harnessed in plastid engineering to solve global challenges, we want to make this chassis accessible to future iGEM teams so that great ideas can be developed. Our wetlab results revolve around the achievement of the first C. reinhardtii chloroplast library in phytobrick standard which, among others, allows to creation of a modular transformation vector for chloroplasts (not previously available in the registry) and the necessary elements to use CRISPR/Cas9 technology.
This section only considers the biological wetlab work, please read about our library-based experimental design which we also consider a result of the summer’s work with the aim of implementing a crispr-cas9 mechanism to achieve homoplasmy in a much quicker way, as necessary for this technology there are important biological containment considerations.
Parts were mainly synthesized through iDT, however there were significant challenges.
Firstly, chloroplast genomes have a high A/T content, therefore many could not be synthesized in this manner. The two solutions were to modify bases in specific regions with high A/T content or add a string of nucleotides at the 5’ and 3’ end which would be later cleaved with a restriction enzyme. We decided on modifying some regions on the sequence (always making sure to maintain the encoded information) as well as yielded some parts from plasmids received from labs working on Chlamydomonas. In the case of CAS9, which was additionally too large, we subdivided the sequence in 4 parts and through PCR added the necessary overhangs for a golden gate assembly without affecting the sequence.
The design of parts included codon-optimization through the software programme developed at Saul Purton lab for chloroplast genes as well as the removal of illegal restriction sites whilst maintaining the integrity of the coding sequence.
Due to a misinterpretation of the novel phytobrick documentation we designed the incorrect overhangs and need to modify each part. We then proceeded to amplify each part with the appropriate overhangs for the phytobrick syntax. New challenges arose with this due to the A/T-rich genome and the high frequency of same-nucleotide tracks. This lead to many sessions of PCR troubleshooting, modulating and tweaking the variables and the PCR protocols to finally achieve all the parts in functional standard.
Yet, for the verification of parts we required a functional plasmid to introduce the gene of interest (GOI), as transgenes are inserted into the chloroplast genome through homologous recombination and there was no available backbone with this purpose in the iGEM registry. We set ourselves the task of developing this plasmid for future teams and to make it modular by providing the homology regions as parts which can be assembled. Following recommendation of Marco Larrea Alvarez from Saul Purton algal biotechnology lab in London we created a promoter and terminator directly linked to homology regions. He has shown interest in these parts for his own research, further, he gave a plasmid to restore photosynthesis in a different strain (TN72) and thus already possessed homology regions into which we could clone a aadA cassette as selection pressure for our GOI. This parallel cloning strategy was the safest way to proceed to achieve verifications.
We verified our parts through sequencing data, indicated on the parts description (for example BBa_K2148001) as well as through restriction digest (for example BBa_K2148005).
Restriction digest was important to verify the correct usage of the phytobrick syntax for cloning into the level 0 backbone. It was particularly useful in the case of cloning the party of homology region with an adjacent promoter (H3R1) which was cloned from plasmids provided to us by Saul Purton. However, due to the size of the fragment we need to extract it in three parts to then assemble by golden gate cloning. When incorporated the part into the level 0 backbone and digested it newly and run on the gel, we saw that the first and second fragments were of the appropriate size but not the third, which was much smaller and we needed to repeat the PCR amplification.
Linköping University
The collaborations with Linköping University has been very helpful for both parties as we exchanged weekly reports through emails and Skype meetings. In depth discussions on the chasis we were working on, Chlamydomonas reinhardtii, were carried out. In addition, we exchanged views on Gibson assembly and other higher level assembly methods' protocols to help each other make an informed decision on the best approach for our project.
