Difference between revisions of "Team:NTU-Singapore/Description"
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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 | + | <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 a medium mutation rate PCR on LDH. The parameters manipulated are the amount of initial DNA template for the PCR. ~400ng of DNA template is used for the medium mutation rate PCR reaction. |
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| − | + | The primers are designed to contain the ATG start codon and TAA stop codons so that the start and stop codons would not be changed. 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 and sent for sequencing. Here are the results. | |
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| − | + | For the first batch, a simpler expression device which only express our mutant LDH is used to measure the growth curve. The anaerobic growth of Shewanella carrying the different LDH mutants were then measured in M1 media for 36 hours. Another second batch of 20 LDH mutants were picked and then ligated infront of a wild type LDP. | |
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| + | Again, the growth of the bacterias carrying the LDH<sup>M</sup>/LDP<sup>WT</sup> operon is then measure under the same conditions. However, the device used for measurement of the second batch was more automated as manual sampling of the first batch is too labour intensive. Please take a look at our protocols. | ||
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Revision as of 16:21, 10 September 2015
Our Project
Ribosomal Binding Site
Introduction
After transcription of a gene, the mRNA will interact with the Ribosome to produce the protein coded in the mRNA in a process called translation. Like how a promoter initiates transcription by allowing RNA polymerase to bind, the ribosome binding site(RBS) provides a platform for ribosomes to bind and positions itself so that translation initiation can occur.
This binding interaction is similar to that of the base pairing of complementary nucleotides. As ribosomes are made out of a highly folded single strand rRNA and small ribosomal proteins, the 3’ end of the 16S rRNA in the 30S subunit has a short nucleotide sequence that complements the mRNA at the RBS. The consensus sequence of the RBS is known as the Shine-Dalgarno sequence, AGGAGG.
The RBS has two purposes in translation initiation, the first being the facilitation of ribosomal binding, the second is to position the ATG start codon of the protein at the peptidyl site of the ribosome. Hence, the distance between the RBS and the start codon plays a pivotal role in translation initiation. However, it’s effect is not explored in our project.
This summer we investigate the effect of substitution mutations on the strength of RBS to initiate translation. As more base pairing interactions would mean a stronger binding affinity to the mRNA, the right substitutions may bring about stronger binding to the RBS and hence, a higher rate of translational initiation.
Our Plan
As Shewanella oneidensis has been an important model organism for the making of the MFC, we would like to characterise a library of RBS mutants in this organism. For this summer, we will mutate a popular RBS, BBa_B0034 from the iGEM registry by making all three possible single base pair substitution for each of the nucleotide in the 12 base pair sequence. For example, switching the first base pair from A to C, G and T. Using eGFP, BBa_E0040 as our reporter, we will characterise the how strong can this RBS initiate translation in Shewanella oneidensis MR1 under a constitutive promoter for this strain, pLac BBa_R011. The final construct for our RBS charecterisation is pLac-RBSM-GFP-TT ligated into pHG101 vector.
We will then characterise these RBS mutants by measuring the GFP fluorescence intensity
