Difference between revisions of "Team:NTU-Singapore/Results"

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<h5>Details and Methods</h5>
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<p class="subtitle explain">We first constructed the measurement construct and ligated it into the pHG101 vector. Then 36 different primers (3 X 12 bp) containing the RBS each with only one different nucleotide are designed to carry out PCR. The kit we used allows the whole plasmid to be replicated and the product can be directly transformed into DH5a. The plasmid is then extracted after overnight cuture and sent for sequencing to confirm sucessfull mutagenesis.
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<p class="subtitle explain">We are lucky to have 35 out of 36 sucessfull mutants. Then the plasmid carrying the mutant RBS is then conjugated into Shewanella.
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<h5>Introduction</h5>
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<h5>Growth curve: batch 1</h5>
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<p class="subtitle explain">Under anaerobic conditions, S. Oneidensis MR1 strain is able to utilise lactate as a energy source via the oxidation of lactate to pyruvate. For this to occur, NAD<sup>+</sup> acting as coenzymes will be reduced, serving as an oxidant. Hence, pyruvate and NADH will be generated in this reaction. The pyruvate can then enter the citric acid cycle and generates more NADH which will then carry the electron to the external environment via the Mtr Pathway.
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Revision as of 17:37, 17 September 2015

NTU SG iGEM 2015




Our Results

Ribosomal Binding Site

He's Brighter :(

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Lactate Metabolism

Zzzapp!!

Read More

Ribosomal Binding Site

Growth Curve

As the measurements are carried out in six batches, the growth of mutants of the same batch are similar but differed a little among batches. This implies that GFP expression does not have any significant effect on the bacteris's growth.

1AT denotes A of base pair 1 is changed to T, the same is applied for other notations of RBS mutants.



GFP Readings

The GFP fluorescence readings of mutants shows interesting results. Although the growth curve is similar among mutants, fluorescence intensities varied among the mutants. In summary, it was found that substitution mutations occurring to the AGGAG sequence within BBa_R0034, AAAGAGGAGAAA, showed a decreased GFP expression while others showed increased GFP output. Especially for mutations to base pair 7, GFP expression is near total-depression.



After normalising the GFP fluorescence readings with the OD600 at T=8, the ratio of the normalised fluorescence of the mutants to wild type RBS is computed and plotted as shown.



We also spotted the mutants on an LB agar plate to have a qualitative view of the GFP brightness. The culture is diluted to OD600 = 0.4 then 3uL of the culture is spotted on to the agar. Brightness of these spots parallels the results in the above graph. For example, 7GA, 8AG and 9GA is the brightest among the mutations on their respective base pairs while 12AC and 11AC are the darkest.