Difference between revisions of "Team:NTU-Singapore/Description"
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| + | At the brainstorming stage of this project, Zhang Lei mentioned that she did some pre-screening of LDH and LDP before. More specifically, her experiments characterized the performance of <i>Shewanella oneidensis</i> MR-1 over-expressing LDH and LDP originated from different bacteria such as <i>Bacillus subtilis</i> and <i>Psedomonas aeruginosa</i>. Characterized in both M1 and M9 medium, the <a href="https://static.igem.org/mediawiki/2015/5/58/Prescreening_of_enzymes.pdf">results</a> indicated that enzymes from <i>Psedomonas aeruginosa</i> work well in the chassis of MR-1, giving a high level of electrical output. | ||
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| + | Considering LDP and LDH are both key players for the lactate metabolism of MR-1, we will mutate the enzyme which would facilitate lactate oxidation to be more efficiently. Hence we can obtain a higher electrical output when these two molecular machines work better. The strategy that we would employ is to randomly make base pair substitutions to the LDH gene so that missense mutations would occur. | ||
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We will carry out error-prone PCR with low fidelity DNA polymerases so that lots of mutants can be generated in a single reaction. The PCR fragments are then ligated to the back of pLac BBa_R0011. We then transform this intermediate construct into DH5a to separate all the mutant fragments. | We will carry out error-prone PCR with low fidelity DNA polymerases so that lots of mutants can be generated in a single reaction. The PCR fragments are then ligated to the back of pLac BBa_R0011. We then transform this intermediate construct into DH5a to separate all the mutant fragments. | ||
Revision as of 17:11, 18 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.
Mutants of RBS BBa_B0034, the original sequence is shown at the top of each column.
We will then characterise these RBS mutants by measuring the GFP fluorescence intensity by using this construct conjugated into Shewanella oneidensis MR1 in the pHG101 vector.
