Team:Cambridge-JIC/Description

Cambridge-JIC

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, 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.

Why Chloroplasts?

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:
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.

Gene Gun

Our complete Do-It-Yourself Gene Gun with all necessary documentation is less than one-hundredth of the commercial price! And it has been firing DNA onto samples!

Growth Facility

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!

Homoplasmy

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!

Project References

  • Mayfield, S. P. (2005); Contribution of 5’ and 3’ untranslated regions of plastid mRNAs to the expression of Chlamydomonas reinhardtii chloroplast genes. Mol Gen Genomics 274: 625–636 DOI 10.1007/s00438-005-0055-y
  • Verma, D., Daniell, H. (2007) Chloroplast Vector Systems for Biotechnology Applications. Plant Physiol. Vol. 145
  • Engler, C., Youles, M., Gruetzner, R., Ehnert, T. M., Werner, S., Jones, J. D., Marillonnet, S. (2014). A golden gate modular cloning toolbox for plants. ACS Synthetic Biology,3(11), 839–843.
  • Krichevsky, A., Meyers, B., Vainstein, A., Maliga, P. and Citovsky, V. (2010) Autoluminescent plants. PLoS ONE 5,e15461.
  • Bock, R (2015). Engineering Plastid Genomes: Methods, Tools, and Applications in Basic Research and Biotechnology. Annual Review of Plant Biology, Vol. 66: 211 -241
  • Jiang W, Brueggeman AJ, Horken KM, Plucinak TM, Weeks DP. (2014) Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii. Eukaryotic Cell. 2014;13(11):1465-1469. doi:10.1128/EC.00213-14.
  • Jin S, Daniell (2015) H. Engineered Chloroplast Genome just got Smarter Trends in plant science. 2015;20(10):622-640. doi:10.1016/j.tplants.2015.07.004.
  • Wannathong, T., Waterhouse, J.C., Young, R.E.B. et al. (2016) New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii. Appl Microbiol Biotechnol 100: 5467. doi:10.1007/s00253-016-7354-6
  • Gangl, Doris; Zedler, Julie A. Z.; Rajakumar, Priscilla D.; Ramos Martinez, Erick Miguel; Riseley, Anthony; Wlodarczyk, Artur Jacek; Purton, Saul; Sakuragi, Yumiko; Howe, Christopher J.; Jensen, Poul Erik; Robinson, Colin. (2015) Biotechnological exploitation of microalgae. Journal of Experimental Botany, Vol. 66, No. 22, 2015, p. 6975-6990.