Team:Exeter/Lab Book

Operon Construction  |  Glycobase

Here we present the lab books for the following work. Please follow the links above or through the brief description below to find out more.

Evolution has provided us with a remarkable variety of polysaccharides that have unique properties and countless uses. Their applications are visible all around us with countless examples in medicine, industry, food, cosmetics and engineering to name a few. The aim of this mini-project was to highlight some of these important polysaccharides; Hyaluronan, Levansucrase and Cyclodextrin.

The purpose of the database was to improve the user friendliness of our product. In the future we envisaged a database containing thousands of enzymes, all expressible in our E.coli, leading to a wealth of sugar production possibilities.

The aim of this mini-project was the construction of single gene plasmids using the glycosyltranseferase (GTase) genes we had synthesised. The gene was to be put behind a promoter and RBS sequence, and in front of a terminator. After construction of these plasmids we wanted to determine the protein expression of the particular genes and further characterise the promoter and terminator when supplemented with appropriate inducers.

The aim of this section of the project was to construct three glycosyltransferase operons. This would be done using two different assembly methods with identical aims; first by Biobrick construction and then by the Gibson assembly method. The purpose if this was for a comparison of the assembly methods intended for future use by iGEM teams. Construction of a complete operon by both methods was attempted with the hope that they yielded the predicted three polysaccharides, synthesised by our chosen sequence of glycosltransferases.

The aim of this mini project was to create a three gene inducible plasmid. Each gene on the plasmid would be controlled by a unique promoter, thereby giving the capability to turn each gene on and off. Why is this important? The ability to turn the genes on and off would allow the creation of a monosaccharide, a disaccharide and a trisaccharide. We hoped that this would provide a proof of concept for a larger model, maybe 100 genes in a genome with the ability to produce a polysaccharide of choice at will.