Difference between revisions of "Team:Cambridge-JIC/Description"

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     <p class="darkBlue" style="font-family:Open Sans; font-size:180%; text-align:center">Improving Chloroplast Transformation Step by Step</p>
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    <p style="font-family:Open Sans; font-size:150%; text-align:center;">Various proteins have already been successfully expressed in chloroplasts, including:</p>
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      <li>monoclonal antibodies
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      <li>growth factors
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    <p style="font-family:Arvo; font-size: 150%; text-align:right">Transgene expression in microalgae can total up to 30-50% of a cell’s dry biomass, as unlike in higher plants and mammals, metabolic energy in microalgae is not directed towards maintaining complex differentiated structures.</p>
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    <p style="font-family:Open Sans; font-size:180%; text-align:center">II. Microalgae chloroplasts as a model for higher plants</p>
 
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     <p style="font-family:Open Sans; font-size:150%; text-align:center;">Research in C. reinhardtii chloroplasts can help achieve the following aims:</p>
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     <p style="font-family:Open Sans; font-size:150%; text-align:center;"><b>Library of Parts</b><br>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.</p>
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      <li>increase yields of oils for biofuels
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      <li>elucidate photosynthetic machinery
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      <li>improve C fixation to combat the international food crisis
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    <p style="font-family:Open Sans; font-size:150%; text-align:center;"><b>Our Improved parts:</b>
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    <br><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2148013">BBa_K2148013</a>
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    <br>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.
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     <p style="font-family:Arvo; font-size: 150%; text-align:right">Due to extensive evolutionary conservation of the chloroplast genome, research in the chloroplasts of microalgae, such as Chlamydomonas reinhardtii, is likely to be applicable to studies of other plants. On the other hand, Chlamydomonas reinhardtii is much easier to grow and maintain than higher plants, such as Marchantia polymorpha. </p>
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    <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2148009">BBa_K2148009</a>
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    <br>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.</p>
 
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    <p class="darkBlue" style="font-family:Open Sans; font-size:180%; text-align:center">III. Bottlenecks of chloroplast engineering
 
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    <p style="font-family:Open Sans; font-size:150%; text-align:center;">Chloroplasts engineering is currently underexplored due to:</p>
 
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      <li>Time issues — it takes 2-3 months to achieve homoplasmy, as state where all chloroplast genome copies have been transformed and when experimental results can be obtained
 
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      <li>Experimental cost — chloroplasts can be reliably transformed almost exclusively by biolistic devices, and commercial biolistic devices are very expensive.
 
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      <li>Lack of modular chloroplast genetic parts available — research is more time-consuming and cumbersome.
 
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    <p class="darkBlue" style="font-family:Open Sans; font-size:180%; text-align:center"> IV. Our solutions</p>
 
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    <p style="font-family:Open Sans; font-size:150%; text-align:center;">Our project aims to tackle most bottlenecks and democratise algal biotechnology:</p>
 
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      <li><a href="https://2016.igem.org/Team:Cambridge-JIC/Parts" style="color:#B7E2F0">Library of chloroplast parts</a> for C. reinhardtii — to facilitate the assembly of synthetic constructs using Phytobricks standard
 
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      <li><a href="https://2016.igem.org/Team:Cambridge-JIC/Biolistics" style="color:#B7E2F0">Open-source biolistics device</a> — for cheaper chloroplasts transformations
 
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      <li><a href="https://2016.igem.org/Team:Cambridge-JIC/GrowthFacility" style="color:#B7E2F0">Open source microalgae growth facility</a> — to growth algae in an affordable way
 
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      <li><a href="https://2016.igem.org/Team:Cambridge-JIC/Homoplasmy" style="color:#B7E2F0">Novel Cas9 strategy</a> — to accelerate the process of achieving homoplasmy
 
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      <li><a href="https://2016.igem.org/Team:Cambridge-JIC/Model" style="color:#B7E2F0">Modelling of Cas9 dynamics</a> — to predict the time our Cas9 stategy would take to transform all copies of a chloroplast’s genome
 
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    <p style="font-family:Open Sans; font-size:180%; text-align:left">References</p>
 
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    <p style="font-family:Open Sans; font-size:150%; text-align:left">1. Wannathong T et al., New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii, Appl Microbiol Biotechnol (2016) 100:5467–5477 </p>
 
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    <p style="font-family:Open Sans; font-size:150%; text-align:left">2. De Las Rivas J et al., Comparative Analysis of Chloroplast Genomes: Functional Annotation, Genome-Based Phylogeny, and Deduced Evolutionary Patterns, Genome Res. 2002. 12: 567-583
 
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    <p style="font-family:Open Sans; font-size:150%; text-align:left">Viitanen P V et al., Metabolic Engineering of the Chloroplast Genome Using the Echerichia coli ubiC Gene Reveals That Chorismate Is a Readily Abundant Plant Precursor for p-Hydroxybenzoic Acid Biosynthesis, Plant Physiol. 2004 Dec; 136(4): 4048–4060.
 
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Revision as of 18:29, 18 October 2016

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 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.