Team:Cambridge-JIC/Description
DESCRIPTION
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!
Why Chlamydomonas?
Chlamydomonas has its natural advantage over many other single celled organisms in the lab such as E.coli or yeast. 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.
In addition, the plant cell systems allows post-transaltional modification such as complex folding and glycosylation.
A well-established model system alga Chlamydomonas with a single chloroplast is a perfect chassis for prototyping plant work in iGEM time scales.
Why Chloroplasts?
Naturally specialized in synthesis
Allows precise editing because DNA integration occurs almost exclusively through homologous integration in Chlamydomonas chloroplast
Higher transgene yields than nucleus
What are the problems?
Complex cloning design
Equipment is expensive and sometimes inaccessible
More time consuming than bacteria engineering
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:
BBa_K2148013
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.
BBa_K2148009
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.
