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

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<h5>Growth curve: batch 2</h5>
 
<h5>Growth curve: batch 2</h5>
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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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<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. Other than that, we used the BioScreen which allows an automated measurements of our cultures in a 96-well plate.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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Growth of Shewanella oneidensis MR1 over expressing the mutant lactate dehydrogenase and the wildtype lactate permease.
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Growth of Shewanella oneidensis MR1 over expressing the mutant lactate dehydrogenase and the wildtype lactate permease.
 
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<h5>Execution</h5>
 
<h5>Execution</h5>
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<p class="subtitle explain">We used GeneMorph error-prone PCR Kit from Agilent Technologies. As we are able to control the frequency of mutations on the PCR products, we carried out four reactions of low mutation rate and medium mutation rate PCR on each gene, LDH and LDP. The parameters manipulated are the amount of initial DNA template for the PCR. For the low mutation rate reaction, a higher amount of initial DNA template is used,~2000ng, to have lower fold amplification. ~400ng of DNA template is used for the medium mutation rate PCR reaction.
 
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<p class="subtitle explain">The primers are designed to contain the ATG start codon and TAA(antisense) stop codon at the 3' end. EcoR1, Xba1 and RBS(BBa_B0034)are designed into the forward primer while Spe1 and Pst1 cut sites are in the reverse primers. With these two primers, the PCR fragments can be digested with Xba1 and Pst1 and ligated in front of pLac promoter in a pSB1AK2 vector digested with Spe1 and Pst1. The ligation products are then transformed into DH5a and plated. 8 colonies are picked for the first bacth of mutant(mLDH/mLDP) and sent for sequencing. Here are the <a>results</a>.
 
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<p class="subtitle explain">The composite of pLac-RBS-mLDH are then ligated infront of a wild type LDP while pLac-RBS-mLDP is ligated infront of a wild type LDH. The resulting expression device would be pLac-RBS-mLDH/mLDP-RBS-(WT)LDP/LDH-TT. This is to ensure that the levels of lactate in the cells could be equalised when two proteins are highly expressed instead of only one of them. This espression device will be ligated into a conjugative plasmid pHG101 and ligated into S. Oneidensis MR1.
 
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<p class="subtitle explain">Then, we measure the growth curve of the MR1 strain containing the pLac-RBS-mLDH/mLDP-RBS-(WT)LDP/LDH-TT construct and compare it with the pLac-RBS-(WT)LDH/LDP-RBS-(WT)LDP/LDH-TT construct.
 
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Revision as of 02:15, 19 September 2015

NTU SG iGEM 2015




Our Results

Ribosomal Binding Site

He's Brighter :(

Read More
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.