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

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<h5>The plan</h5>
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<h5>Growth curve: batch 2</h5>
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<p class="subtitle explain">As LDP and LDH are key players for the energetics of the bacteria, we are trying to make mutants of the two enzymes which will transport and catalyse more efficiently than the wild type version. Hence, we can obtain a higher electrical output when these two molecular machines work better. The stratergy that we would employ is to randomly make single base pair substitutions to both genes so that missense mutations would occur. When the catalytic residues are altered, the effects on the activity of protein can go two ways, either having an increased or a decreased catalytic activity.
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<p class="subtitle explain">We did another round of measurements with additional mutants. However, for the second batch, our lactate dehydrogenase mutants were now ligated to the wildtype version of lactate permease. For lactate permease mutants, we did not carry on further as multiple ligations lead to failure.
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<div align="centre"><img class="" src="https://static.igem.org/mediawiki/2015/7/78/SH8_mutants.png" width="600px" height="450px"></div><p style="text-align:center; font-size: 19px; color: black">
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Growth of Shewanella oneidensis MR1 over expressing lactate permease.
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<p style="text-align:left; font-size:20px">We carried out error-prone PCR with low fidelity DNA polymerases on the two genes so that lots of mutants can be generated in a single reaction. We then transform E. Coli with the PCR fragments to separate all the mutant fragments and also for cloning purposes.
 
 
 
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Revision as of 01:56, 19 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.