Team:Edinburgh/Results

From 2008.igem.org

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Latest revision as of 02:00, 30 October 2008

Results and current status as of 29 October 2008. We are hoping to obtain some last-minute results to present at the Jamboree.

Overview

Our results are promising but our dream of using bacteria to convert cellulose into starch and β-carotene is still unrealised. Fortunately, the work will be continued by two undergraduate students after Christmas.

Cellulolysis device

cenA (endoglucanase), cex (exoglucanase) and bglX (beta glucosidase): BioBricks^TM made and ribosome binding site added; ready to test. Last minute result: possible cenA and bglX activity detected using chromogenic substrate, but needs to be repeated and confirmed: hope for more results in our poster and talk at the Jamboree!
P_cstA (promoter): BioBrick^TM made and tested - successful.
- P_cstA promoter assay with xylE as reporter

Glycogenesis device

glgC and glgC16 (ADP-glucose pyrophosphorylase, responsible for rate limiting step in glycogenesis): BioBricks^TM made and tested - successful.

Starch synthesis device

SU1 and ISO2 (isoamylases): Work in progress - pre-BioBrick^TM constructs in pSB1A2 have been made using the BABEL protocol, but it is still necessary to remove internal EcoRI sites before fully compliant BioBricks^TM will be ready to test. This work will be continued in an undergraduate honours project.

Lycopene generator

dxs+crtE+crtB+crtI+crtY: Work in progress - encountered problem at final step. Assembly of the three genes crtE, crtB and crtI together in one construct has failed so consistently and in so many unexpected ways that we are starting to wonder whether this combination is toxic to our host strain under the growth conditions used, though there seems no obvious reason to expect this. Further experiments are required.

Limonene synthase device

dxs+LIMS1+appY: BioBrick^TM made - still being tested. A GC/MS test of an ethyl acetate extract from an induced culture did not detect limonene, but the extraction procedure still needs to be properly validated, so we still can't be sure whether the limonene synthase is working in our host.

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+Our results are promising but our dream of using bacteria to convert cellulose into starch and β-carotene is still unrealised. Fortunately, the work will be continued by two undergraduate students after Christmas.
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+* '''''cenA''''' (endoglucanase), '''''cex''''' (exoglucanase) and '''''bglX''''' (beta glucosidase): BioBricks<sup>TM</sup> made and ribosome binding site added; ready to test. Last minute result: possible ''cenA'' and ''bglX'' activity detected using chromogenic substrate, but needs to be repeated and confirmed: hope for more results in our poster and talk at the Jamboree!
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+** [[Team:Edinbrugh/Results/PcstA-xylE|P<sub>''cstA''</sub> promoter assay with ''xylE'' as reporter]]
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+* '''''glgC''''' and '''''glgC16''''' (ADP-glucose pyrophosphorylase, responsible for rate limiting step in glycogenesis): BioBricks<sup>TM</sup> made and tested - successful.
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+* '''''SU1''''' and '''''ISO2''''' (isoamylases): Work in progress - pre-BioBrick<sup>TM</sup> constructs in pSB1A2 have been made using the BABEL protocol, but it is still necessary to remove internal EcoRI sites before fully compliant BioBricks<sup>TM</sup> will be ready to test. This work will be continued in an undergraduate honours project.
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+* '''''dxs+crtE+crtB+crtI+crtY''''': Work in progress - encountered problem at final step. Assembly of the three genes ''crtE, crtB'' and ''crtI'' together in one construct has failed so consistently and in so many unexpected ways that we are starting to wonder whether this combination is toxic to our host strain under the growth conditions used, though there seems no obvious reason to expect this. Further experiments are required.
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+* '''''dxs''+''LIMS1''+''appY''''': BioBrick<sup>TM</sup> made - still being tested. A GC/MS test of an ethyl acetate extract from an induced culture did not detect limonene, but the extraction procedure still needs to be properly validated, so we still can't be sure whether the limonene synthase is working in our host.
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-<li><a href="https://2008.igem.org/Team:Edinburgh" title="Home"><span>Home</span></a></li>
-<li><a href="https://2008.igem.org/Team:Edinburgh/Project" title="Project" rel="dropmenu1_a"><span>The Project</span></a></li>
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-<a href="https://2008.igem.org/Team:Edinburgh/Team">Overview</a>
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-== Glycogen Assay ==
-In order to determine whether our mutation (G336E) has increased the yield of glycogen in the cells we had to come up with a method of assaying glycogen production. The one we designed simply involves adding Gram's iodine solution dropwise on to colonies grown on nutrient agar. The test works by the iodine solution staining the glycogen resulting in a brown colour: a cell containing more glycogen will stain darker brown than a cell containing less glycogen.
-In order to test this assay we grew ''E. coli'' on two different media, one which would encourage glycogen production and a second that would not. It has been documented that glycogen production is increased in ''E. coli'' cells by growth in a medium rich in a carbon source, so we made up the following plates:
-# Nutrient agar
-# Nutrient agar + 2% glucose
-Results of the staining of colonies on these two plates are as follows:
-[[Image:Edinburgh=Glycogen-Assay.jpg‎]]
-This shows that the stain colony on the (+)glucose plate (a) is darker than the stained colony on the (-)glucose plate (b), indicating that the colony on the (+)glucose plate contains more glycogen than that on the (-)glucose plate. (These results were apparent after ~5 mins of staining. After ~30 mins of staining the colour began to diminish until no apparent difference could be seen between the two colonies, as can also be seen by the colony to the left of the circled colony on each plate in the figure)