We have built a toolbox for chloroplast transformation. In our chosen organism Chlamydomonas reinhardtii (Chlamy) we have targeted all the transformation steps and improved each of them. We have built a library of tested parts optimised for Chlamydomonas or related chloroplasts. We built a gene gun which is less than 1/100 of commercial price and an incubator for our cells with many functions. Moreover we modelled the behaviour of transformed systems. Finally we designed a wetlab tool which could help achieve essential homoplasmy (transformation of all copies of chloroplast DNA) in one generation instead of 2-3 months of selection. Our design incorporated numerous discussions about biosafety and our library contains the relevant parts for practical implementation!
Chlamydomonas, a single-celled alga with only one chloroplast, has its natural advantage over many other single-celled organisms in the lab such as E.coli and yeast. It is a perfect chassis for prototyping plant work in iGEM time scales.
Unlike E. coli or yeast it does not need extra carbon source such as glucose. This will make a huge difference in an industrial scale.
The plant cell systems allows post-translational modification such as complex folding and glycosylation.
Higher transgene yields than nucleus
Allows precise editing because DNA integration occurs almost exclusively through homologous integration in Chlamydomonas chloroplast
Naturally specialized in synthesis
What are the problems?
Complex cloning design
Equipment is expensive and sometimes inaccessible
More time-consuming than bacteria engineering
Multiple plastid genomes to transform
Improving Chloroplast Transformation Step by Step
Library of Parts
Our library of parts contains many parts necessary for synthetic biology of chloroplasts. They have been tested by cloning in E. coli, some by shooting into Chlamy and extracting, some even by sequencing. They all were designed with the intention to facilitate bringing our homoplasmy tool into practice.
Our Improved parts:
Cas9 is a molecular tool which together with its guide RNA can cut DNA sequences at sequence-specific places. Our Cas9 is codon-optimized for Chlamydomonas reinhardtii chloroplast chassis (likely useable in other chloroplasts). Additionally it has a fusion tag to link reporter genes such as fluorescent proteins. It is fully compatible with the increasingly popular Phytobrick standard.
GFP is the most classic fluorescent reporter protein with very wide range of usage. Our GFP is again codon-optimised for Chlamydomonas reinhardtii chloroplast. Moreover it is compatible with Phytobrick standard.
Always wanted to have a portable controlled environment for your Petri dishes full of organisms which can even Tweet pictures of itself? We have made one! It has various functions, is cheap, well documented and again DIY. This can really bring algae into small and community labs and make synthetic biology accessible and understandable to public!
Our ultimate wetlab goal was to build a very modular device based on CRISPR/Cas9 which would accelerate the post-transformation process. To get your genes into all the copies of chloroplast DNA has never been so quick! As we are pioneers in using plant and algal chloroplasts in iGEM, our tool is theoretical but well thought through (see our Notebook for details), incorporating all environmental safety advice from scientists and the library of parts is designed specifically to facilitate this to happen in real!
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