Team:NRP-UEA-Norwich/BioBricks
BIOBRICKS
In order to assess whether our BioBricks would function in our model organism Shewanella oneidensis MR-1 we attempted to transform and express two BioBricks into S. oneidensis MR-1; J04450 containing a gene for the red fluorescent protein (RFP) and K584001 which contains a gene for the green fluorescent protein (GFP). These BioBricks were chosen as they contain reporter genes and successfully transformed colonies could be easily identified under UV light.
As S. oneidensis MR-1 are not able to undergo heat shock transformation we used a tri-parental conjugation approach (see protocol). Parent Escherichia coli strains contained J04450 and K584001 respectively using a heat shock approach according to protocol. For step 9 in the tri-parental conjugation protocol chloramphenicol was used instead of kanamycin as BioBricks contain chloramphenicol resistance as a selective marker instead of kanamycin.
After serial dilutions for all attempted conjugations no individual S. oneidensis MR-1 colonies survived, and therefore the cells failed to display chloramphenicol resistance. Escherichia coli parent and helper strains survived, so carbenicillin resistance could have been transferred from S. oneidensis MR-1 to E. coli. Also possible however is that E. coli developed spontaneous carbenicillin resistance as they can easily gain mutations in the carbenicillin target protein, inferring resistance.
In the future iGEM teams should investigate this to confirm the origin of replication is in fact the issue with BioBrick expression in S. oneidensis MR-1. If this is in fact the case future iGEM teams can then move on to apply synthetic biology to optimise the origin of replication of some BioBricks for the highest possible copy number in S. oneidensis MR-1. These can then serve as a backbone for the cloning of further genes to be expressed in S. oneidensis MR-1. This exercise will compound in value as this organism becomes more widely used for synthetic biology.
In the future iGEM teams should investigate this to confirm the origin of replication is in fact the issue with BioBrick expression in S. oneidensis MR-1. If this is the case future iGEM teams can then move on to apply synthetic biology to optimise the origin of replication of some BioBricks for the highest possible copy number in S. oneidensis MR-1. These can then serve as a backbone for the cloning of further genes to be expressed in S. oneidensis MR-1 allowing more detailed investigations into the biotech applications of this organism.
https://2008.igem.org/Team:Harvard
https://2015.igem.org/Team:NTU-Singapore
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