Team:Exeter/Results

The 3-Gene Inducible Plasmid  |  Single Gene Plasmids and Enzyme Characterisation

Result in Brief:

We created a database containing over 120 different glycosyltransferases found mainly in E. coli. The glycosyltransferases were listed according to their donor sugar, acceptor sugar, organism, structure and whether they were previously characterised or not. From the database, the Exeter iGEM team could then see the possible repeat unit chains (and therefore polysaccharides) which could be produced based on the glycosyltransferases available. GlycoBase became an important part of the iGEM project, acting as the mediator between the wet lab and dry lab teams.

Result in Brief:

Result in Brief:

Taking on the challenge of designing and constructing an operon in such a short experimental window was always expected to be ambitious, and running two different techniques side by side meant that the project ran out of time. The allotted time was insufficient to finish either BioBrick assembly or Gibson assembly of an entire operon, and consequently it was impossible to test for biosynthesis of a novel polysaccharide. Through BioBrick assembly, several RBS-gene constructs were made which, the promoter_RBS construct was made and terminators were added to the ends of the constructs Some of these we have managed to transfer into pSB1C3 plasmids and have gone on to the registry as new BioBricks. The use of both Gibson assembly and BioBrick assembly has built a comparative picture of the two assembly methods in the hope that future iGEM teams might get quantitative data to assist decisions over the use of Gibson and BioBrick assembly in the iGEM competition. We hope that if we have a chance to continue this work, we could complete one of the operons and test for novel polysaccharides.

Result in Brief:

Due to an insufficient experimental time window, we were unable to complete the ambitious attempt to assemble an entire operon whilst comparing Bio-Brick Assembly and Gibson Assembly, as noted above. Consequently it was impossible to test for biosynthesis of a novel polysaccharide. We hope that if we have a chance to continue this work, we could complete one of the operons and test for novel polysaccharides.

Result in Brief:

The unique and variable properties of polysaccharides in nature led us to undertake cloning and producing a variety of polysaccharides from different natural sources into our E.coli. These polysaccharides have variable structures and important applications in diverse business sectors. The Hyaluronan synthase and cyclodextrin glycosyltransferase enzymes were successfully cloned and sequenced inside pSB1C3 plasmids and submitted to the registry for future teams to realise their potential products (BBa_K764022 and BBa_K764026 respectively). Each of the showcase enzymes were successfully linked to terminators, sequenced and submitted to the registry, including levansucrase. Levansucrase was an adaption of Newcastle iGEM 2010’s gene for SacB, adding a terminator on for more ease of use for future teams as they can by-pass this cloning step. (NEED ALEX C TO INFORM ME WHERE THE LAB GOT FROM HERE). Unfortunately due to time restraints we were unable to produce our desired polysaccharide in our E. coli, leaving an opportunity for a future iGEM team to continue our work.

Result in Brief:

With: Ryan Edginton, Alice Bond, James Lynch and Liam Stubbington

Result in Brief:

We set out to create a three gene inducible plasmid and nearly got there. At the end of the project we managed to add two genes to promoter, RBS and terminators and create the promoter_RBS and the gene terminator ready to put together for the third construct. A ligation of the two full constructs is in the freezer ready to be transformed. Although the project has come short and the goal has not been achieved, we were still able to use the constructs made to test for protein expression to help characterise the promoters we used. Given more time it would be great to be able to complete this side of the project in the future seeing as we managed to get so close.

Result in Brief:

Overall, whilst it was not possible to conduct the enzyme assays or undertake mass spectrometry which were important, this mini-project accomplished the significant cloning work required to undertake such experiments in the future. If time was not an issue, then all glycosyltransferases would be assayed multiple times to determine prinicipally Vmax and KM values to help with the modelling of our system, which in turn would be used to optimise the GlycoBase database. Mass spectrometry would be used to identify both oligosaccharide production and loss of diphosphonucleotide carrier. SDS-PAGE would finally confirm functionality and molecular weight of our glycosyltransferase proteins.