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:

A URL was created for GlycoBase, which was uploaded to form the online interface GlycoWeb. In GlycoWeb we have created a piece of software which not only allows users to search a polysaccharide and see if its creation is possible, but also to add to the database interactively based on their own research. This will lead to better glycosylytransferase characterisation in the future. GlycoWeb is an open source piece of software. GlycoApp, the android software version of GlycoBase is currently under construction. In the future we see Material Scientists, Engineers and Biologists ordering their desired polysaccharide using GlycoApp, therefore improving the efficiency and ease of desired material creation.

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. Several BioBricks, both coding and composite parts, were created for the operon. While they were not combined to make the final construct, they will go to the registry and hopefully be used by future teams for polysaccharide production and the continuation of our work.

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:

The motivation for constructing a mathematical model to represent our system consisted of two parts. Firstly, to provide additional software to aid GlycoWeb. The model that has been made can rank out puts of database giving the user optimal enzyme combinations that facilitate the build of a given polysaccharide. This is achieved by using a ranking system, where the rank of a combination of enzymes is dependant on the yield and rate of production of the desired polysaccharide Whilst the model is currently uncalibrated, the software is ready to be united with GlycoWeb in this way.

The second motivation behind the modelling task may not have an instant and obvious application objective, but some would argue that it is a far more important reason to invest in a piece of science. The model was intended to represent each reaction inside the cell mathematical and precisely. Upon alignment with data, the model could highlight fundamental properties of the system we use. It could indicate assumptions which we held true about the system to be false and gives an abstract view of the biology which we are working with.

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 V(sub)max(/sub) and K(sub)M(/sub) 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. The mini-project sufficed in submitting the majority of single gene plasmids (BBa_K764000-BBa_764004, BBa_K754007, BBa_K764022 and BBa_K764023) in addition to a number of constructs (BBa_K764009, BBa_K764011, BBa_K764024, BBa_K764026, BBa_K764033 and BBa_K764034) to the Parts Registry. Descriptions are avaialable on Bio-Bricks.