Team:UEA-JIC Norwich/Nittygritty-algae

From 2011.igem.org

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[[File:Algae_banister.jpg|center|900x89px]]
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<h1><p style=""background-color:#000000">Algae</h1>
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<h1 style="font-family:verdana;color:Green">ALGAE</h1>
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<p>We are planning to transform the algal species <i>Chlamydomonas reinhardtii</i>. This is a single celled, photosynthetic eukaryote. It is easily transformable, either by: electroporation; the bacterium Agrobacterium tumorfaciens; glass beads; or by the use of a biolistic particle delivery system (gene gun). We will be using the specific strain CC-4350 cw15-302 mt+. This strain is biflagellate with a high transformation frequency, due in part to its lack of a cell wall. Its genome has been sequenced, allowing us to research its codon bias.</p>
+
<p>We have preformed two different types of protocol; glass bead and electroporation to transform the algal species <i>Chlamydomonas reinhardtii</i>. This is a single celled, photosynthetic eukaryote. We have been using the specific strain CC-4350 cw15-302 mt+. This strain is biflagellate with a high transformation frequency, due in part to its lack of a cell wall. Its genome has been sequenced, allowing us to research its codon bias.</p>
-
<p>There are twenty common amino acids, but over sixty codon configurations. So, each amino acid can be coded for by multiple codons. The code is thus said to be degenerate. However, most organisms display a preference for one codon or another, and so prefer to express a given amino acid by a certain codon. This is known as the codon bias. We researched the codon bias for <i>Chlamydomonas reinhardtii</i>, and plan to use this information to optimise the Biobricks we wish to use for expression in <i>C. reinhardtii</i>.
+
<p>There are twenty common amino acids, but over sixty codon configurations. So, each amino acid can be coded for by multiple codons. The code is thus said to be degenerate. However, most organisms display a preference for one codon or another, and so prefer to express a given amino acid by a certain codon. This is known as the codon bias. We researched the codon bias for <i>Chlamydomonas reinhardtii</i>, and are using this information to optimise the Biobricks we wish to use for expression in <i>C. reinhardtii</i>.</p>
 +
<br>
 +
<h2 style="font-family:verdana;color:green">Chlamydomonas Fact File</h2>
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<p>Below is the plasmid we designed for the algal construct. We used the Ble gene conferring the Bleomycin resistance cassette for the selection marker. This confers resistance to the Bleomycin family of antibiotics (we used phleomycin to select our transformed cells). One other option we considered was selection by Arginine, but this would narrow down the range of possible algal species future iGEM teams could use, as a strain with a mutated Arginine Biosynthesis gene would have to be used.</p>
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<p>Name:<i>Chlamydomonas reinhardtii</i></p>
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</html>
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<br>
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[[File:Circular_plasmid_algae.jpg]]
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<h3 style="font-family:verdana;color:green">Attributes</h3>
 +
<p>Can be transformed by a variety of methods – electroporation; the bacterium <i>Agrobacterium tumorfaciens</i>; glass beads; or by a biolistic particle delivery system (gene gun)</p>
 +
<p>Eukaryotic photosynthetic organism – therefore its post translational modifications will more closely reflect those seen in plants and other higher organisms when compared to, for example, <i>E.coli</i></p>
 +
<br>
 +
<h3 style="font-family:verdana;color:green">Difficulties of Use</h3>
 +
<p>Many strains have a cell wall, and therefore prove difficult to transform</p>
 +
<p>Growing time of around a week in cultures or plates</p>
 +
<p>Low transformation frequency due to genome integration of plasmids</p>
 +
<br>
 +
<h2 style="font-family:verdana;color:green">Growth Conditions</h2>
 +
<p>Requires TAP (tris-acetone phosphate) media</p>
 +
<p>Must be grown in sunlight</p>
 +
<p>Must be grown at 25°C</p>
 +
<p>Must be grown in an incubator shaker to ensure adequate aeration of the media with carbon dioxide</p>
 +
 
 +
 
 +
 
 +
<table border="1">
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<tr>
 +
<th>Stock solution</th>
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<th>Volume</th>
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<th>Component</th>
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<th>Concentration in stock Solution</th>
 +
<th>Concentration in final media</th>
 +
</tr>
 +
<tr>
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<td>Tris base</td>
 +
<td>2.42g</td>
 +
<td>H2NC(CH<sub>2</sub>OH)<sub>3</sub> </td>
 +
<td></td>
 +
<td>2.00 . 10<sup>-2</sup>M</td>
 +
</tr>
 +
<tr>
 +
<td>TAP-salts (Beijerinck salts)</td>
 +
<td>25mL</td>
 +
<td>NH<sub>4</sub>Cl MgSO<sub>4</sub> . 7H<sub>2</sub>O CaCl<sub>2</sub> . 2H<sub>2</sub>O</td>
 +
<td>15 g . L<sup>-1</sup>
 +
4 g . L<sup>-1</sup>
 +
2 g . L<sup>-1</sup></td>
 +
<td>7.00 . 10<sup>-3</sup> M
 +
8.30 . 10<sup>-4</sup> M
 +
4.50 . 10-4 M</td>
 +
</tr>
 +
<tr>
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<td>Phosphate solution</td>
 +
<td>1mL</td>
 +
<td>K<sub>2</sub>HPO<sub>4</sub>
 +
KH<sub>2</sub>PO<sub>4</sub></td>
 +
<td>28.8 g . 100 mL<sup>-1</sup>
 +
14.4 g . 100 mL<sup>-1</sup></td>
 +
<td>1.65 . 10<sup>-3</sup> M
 +
1.05 . 10<sup>-3</sup>M</td>
 +
</tr>
 +
<tr>
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<td>Hunter’s trace Elements</td>
 +
<td>1mL</td>
 +
<td>5.00 g . 100 mL<sup>-1</sup>
 +
2.20 g . 100 mL<sup>-1</sup>
 +
 
 +
1.14 g . 100 mL<sup>-1</sup>
 +
 
 +
0.50 g . 100 mL<sup>-1</sup>
 +
 
 +
0.50 g . 100 mL<sup>-1</sup>
 +
 
 +
0.16 g . 100 mL<sup>-1</sup>
 +
 
 +
0.16 g . 100 mL<sup>-1</sup>
 +
 
 +
0.11 g . 100 mL<sup>-1</sup></td>
 +
<td>5.00 g . 100 mL<sup>-1</sup>
 +
2.20 g . 100 mL<sup>-1</sup>
 +
 
 +
1.14 g . 100 mL<sup>-1</sup>
 +
 
 +
0.50 g . 100 mL<sup>-1</sup>
 +
 
 +
0.50 g . 100 mL<sup>-1</sup>
 +
 
 +
0.16 g . 100 mL<sup>-1</sup>
 +
 
 +
0.16 g . 100 mL<sup>-1</sup>
 +
 
 +
0.11 g . 100 mL<sup>-1</sup></td>
 +
<td>1.34 . 10<sup>-4</sup> M
 +
1.36 . 10<sup>-4</sup> M
 +
1.84 . 10<sup>-4</sup> M
 +
4.00 . 10<sup>-5</sup> M
 +
3.29 . 10<sup>-5</sup> M
 +
1.23 . 10<sup>-5</sup> M
 +
1.00 . 10<sup>-5</sup> M
 +
4.44 . 10<sup>-6</sup> M</td>
 +
</tr>
 +
<tr>
 +
<td>Acetic Acid</td>
 +
<td>1mL</td>
 +
<td>CH<sub>3</sub>COOH</td>
 +
<td></td>
 +
<td></td>
 +
</tr>
 +
</table>
 +
 
 +
<h3 style="font-family:verdana;color:green">Method:</h3>
 +
 
 +
<p>Dissolve Na<sub>2</sub>EDTA . 2H<sub>2</sub>O in 100 mL dd-H<sub>2</sub>O by heating to 60-80 °C, then adjust pH with KOH to 5.0.
 +
Add all trace elements separately and check the pH value constantly. The pH value should not increase
 +
above 6.8, otherwise MnSO<sub>4</sub> may precipitate. Let the solution stand at 4 °C; when the colour changes
 +
from orange to red after approx. 2 weeks, filter it, split it and store at -20 °C teflon or polycarbonate
 +
containers (do not use glass containers for trace elements as these tend to adsorb to the glass surface).
 +
After addition of acetic acid the pH should range at about 7.0.</p>
 +
 
 +
<p>Adjust medium to final pH of 6.0 or as desired with acetic acid and autoclave at 121 °C for 20
 +
min.</p>
 +
 
 +
<p>For stock cultures on agar slants add 1.0-1.3 % Agar (e.g. purified high strength, 1000 g . cm-²) to
 +
prepared medium before autoclaving.</p>
 +
The genome integration of plasmids lowers the transformation frequency of <i>Chlamydomonas</i>, as it is difficult to direct the specific site the DNA will integrate into. This raises the risk of integration within a gene vital for the organisms survival, thus killing the organism. The presence of a cell wall potentially hinders the transformations of many strains, but strains are available that are cell wall deficient (such as the CC-4350 cw15-302 mt+ strain we are using). Using such a strain negates the problem.
 +
 
 +
<h3 style="font-family:verdana;color:green">Plasmid Design:</h3>
 +
<p>The genome integration of plasmids lowers the transformation frequency of <i>Chlamydomonas</i>, as it is difficult to direct the specific site the DNA will integrate into. This raises the risk of integration within a gene vital for the organisms survival, thus killing the organism. The presence of a cell wall potentially hinders the transformations of many strains, but strains are available that are cell wall deficient (such as the CC-4350 cw15-302 mt+ strain we are using). Using such a strain negates the problem.</p>
 +
<br>
 +
References:  
 +
<br>
 +
<br>
 +
1. http://cccryo.fraunhofer.de/sources/files/medien/TAP.pdf
 +
<br>
 +
<br>
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<body>
 
-
<p>The Ble gene we used came from the Chlamydomonas Centre (USA). This gene has been adapted for use in eukaryotic organisms, including the insertion of two introns (see above) and the addition of a 5' and 3' untranslated region (UTR). We used PCR (polymerase chain reaction) to extract the gene from the plasmid it came in, simultaneously adding the iGEm prefix and suffix to either end. We encountered difficulties in using the Ble gene, in that it contained an illegal Xba1 site in the 3' UTR. Adhering to the Biobrick assembly standards was very important to us, as we wished to make the accessibility of these two species as easy as possible for future teams. Therefore we plan to use site directed mutagenesis to remove this site.</p>
 
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Latest revision as of 20:28, 21 September 2011

University of East Anglia-JIC

UNIVERSITY OF EAST ANGLIA-JOHN INNES CENTRE


Algae banister.jpg

ALGAE

We have preformed two different types of protocol; glass bead and electroporation to transform the algal species Chlamydomonas reinhardtii. This is a single celled, photosynthetic eukaryote. We have been using the specific strain CC-4350 cw15-302 mt+. This strain is biflagellate with a high transformation frequency, due in part to its lack of a cell wall. Its genome has been sequenced, allowing us to research its codon bias.

There are twenty common amino acids, but over sixty codon configurations. So, each amino acid can be coded for by multiple codons. The code is thus said to be degenerate. However, most organisms display a preference for one codon or another, and so prefer to express a given amino acid by a certain codon. This is known as the codon bias. We researched the codon bias for Chlamydomonas reinhardtii, and are using this information to optimise the Biobricks we wish to use for expression in C. reinhardtii.


Chlamydomonas Fact File

Name:Chlamydomonas reinhardtii


Attributes

Can be transformed by a variety of methods – electroporation; the bacterium Agrobacterium tumorfaciens; glass beads; or by a biolistic particle delivery system (gene gun)

Eukaryotic photosynthetic organism – therefore its post translational modifications will more closely reflect those seen in plants and other higher organisms when compared to, for example, E.coli


Difficulties of Use

Many strains have a cell wall, and therefore prove difficult to transform

Growing time of around a week in cultures or plates

Low transformation frequency due to genome integration of plasmids


Growth Conditions

Requires TAP (tris-acetone phosphate) media

Must be grown in sunlight

Must be grown at 25°C

Must be grown in an incubator shaker to ensure adequate aeration of the media with carbon dioxide

Stock solution Volume Component Concentration in stock Solution Concentration in final media
Tris base 2.42g H2NC(CH2OH)3 2.00 . 10-2M
TAP-salts (Beijerinck salts) 25mL NH4Cl MgSO4 . 7H2O CaCl2 . 2H2O 15 g . L-1 4 g . L-1 2 g . L-1 7.00 . 10-3 M 8.30 . 10-4 M 4.50 . 10-4 M
Phosphate solution 1mL K2HPO4 KH2PO4 28.8 g . 100 mL-1 14.4 g . 100 mL-1 1.65 . 10-3 M 1.05 . 10-3M
Hunter’s trace Elements 1mL 5.00 g . 100 mL-1 2.20 g . 100 mL-1 1.14 g . 100 mL-1 0.50 g . 100 mL-1 0.50 g . 100 mL-1 0.16 g . 100 mL-1 0.16 g . 100 mL-1 0.11 g . 100 mL-1 5.00 g . 100 mL-1 2.20 g . 100 mL-1 1.14 g . 100 mL-1 0.50 g . 100 mL-1 0.50 g . 100 mL-1 0.16 g . 100 mL-1 0.16 g . 100 mL-1 0.11 g . 100 mL-1 1.34 . 10-4 M 1.36 . 10-4 M 1.84 . 10-4 M 4.00 . 10-5 M 3.29 . 10-5 M 1.23 . 10-5 M 1.00 . 10-5 M 4.44 . 10-6 M
Acetic Acid 1mL CH3COOH

Method:

Dissolve Na2EDTA . 2H2O in 100 mL dd-H2O by heating to 60-80 °C, then adjust pH with KOH to 5.0. Add all trace elements separately and check the pH value constantly. The pH value should not increase above 6.8, otherwise MnSO4 may precipitate. Let the solution stand at 4 °C; when the colour changes from orange to red after approx. 2 weeks, filter it, split it and store at -20 °C teflon or polycarbonate containers (do not use glass containers for trace elements as these tend to adsorb to the glass surface). After addition of acetic acid the pH should range at about 7.0.

Adjust medium to final pH of 6.0 or as desired with acetic acid and autoclave at 121 °C for 20 min.

For stock cultures on agar slants add 1.0-1.3 % Agar (e.g. purified high strength, 1000 g . cm-²) to prepared medium before autoclaving.

The genome integration of plasmids lowers the transformation frequency of Chlamydomonas, as it is difficult to direct the specific site the DNA will integrate into. This raises the risk of integration within a gene vital for the organisms survival, thus killing the organism. The presence of a cell wall potentially hinders the transformations of many strains, but strains are available that are cell wall deficient (such as the CC-4350 cw15-302 mt+ strain we are using). Using such a strain negates the problem.

Plasmid Design:

The genome integration of plasmids lowers the transformation frequency of Chlamydomonas, as it is difficult to direct the specific site the DNA will integrate into. This raises the risk of integration within a gene vital for the organisms survival, thus killing the organism. The presence of a cell wall potentially hinders the transformations of many strains, but strains are available that are cell wall deficient (such as the CC-4350 cw15-302 mt+ strain we are using). Using such a strain negates the problem.


References:

1. http://cccryo.fraunhofer.de/sources/files/medien/TAP.pdf

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