Difference between revisions of "Team:NTU-Singapore/Description"
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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. | ||
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| − | After that we will complete the intermediate construct into an operon consisting of the mutant LDH and the wild type LDP, using BBa_B0015 as our transcription terminator and BBa_R0034 as our RBS. After transferring our operon into the pHG101 backbone, similar methods will be employed to conjugate the plasmid into Shewanella. Finally, we will measure the growth curves of the bacteria for carrying each mutant LDH under anaerobic conditions. <img src="https://static.igem.org/mediawiki/2015/3/3d/Sh78.png" | + | After that we will complete the intermediate construct into an operon consisting of the mutant LDH and the wild type LDP, using BBa_B0015 as our transcription terminator and BBa_R0034 as our RBS. After transferring our operon into the pHG101 backbone, similar methods will be employed to conjugate the plasmid into Shewanella. Finally, we will measure the growth curves of the bacteria for carrying each mutant LDH under anaerobic conditions.<br> <br><div align="centre"><img class="" src="https://static.igem.org/mediawiki/2015/3/3d/Sh78.png" width="831.6px" height="154.6px"></div></p> |
Revision as of 15:51, 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
