Team:Exeter/Lab Book

Operon Construction  |  Glycobase

Contributors: Alice Bond and Liam Stubbington

The aim of this mini-project will be the construction of single gene plasmids both individually and with promoters and terminators, that will ultimately be sent as BioBricks to the registry. Single gene expression plasmids will be constructed and, time permitting, measure gene expression and validate the functioning of the promoter and terminator when supplemented with appropriate inducers.

Enzyme characterisation will be significant in this project in two ways. Firstly, GTases are very poorly understood and of the 128 GTases identified and known to exist in the production of O-antigens of all the E.coli strains, only 20 have been characterised through published literature. Secondly, verification of the preferred substrate and terminal acceptor saccharide in addition to enzyme kinetics will be essential for the proper functioning of the dry lab database. To determine enzyme kinetics, a glycosyltransferase assay will be used, based on the cleavage of inorganic phosphate from the pyrophosphate moiety of the sugar diphosphonucleotide carrier. The release of inorganic phosphate yields a colour change which can be detected simply using a spectrophotometer. Because release of the diphosphonucleotide carrier is quantitative to enzyme rate, the change in colour will reflect enzyme catalysis rate directly. In addition, SDS-PAGE will be used to determine the molecular weight of each GTase and compared to predicted values, as well as solubility of each GTase to check functionality in vivo.

Evolution has provided us with a remarkable variety of polysaccharides that have unique properties and countless uses. Their applications in medicine, industry, food, cosmetics, engineering etc. have been recognised and the biological synthesis of these polysaccharides has begun.

Hyaluronan is a powerful lubricant found in human joints and skin and so has medical applications from surgical glues to skin moisturisers. Expression of Levansucrase outside the cell by the addition of a signal peptide ompA, means we are able to avoid the toxic properties that levansucrose has inside the cell, demonstrate our polysaccharides can be produced extracellularly also and show that we can produce levansucrose with our E. coli. Made from linking fructosyl units together, Levansucrose is a potential glue recognised by Newcastle iGEM 2010 (BacillaFilla) that could repair cracks in concrete. The third polysaccharide we are producing is a cyclic oligosaccharide made from starch rather than a linear polysaccharide; cyclodextrin. Cyclodextrin has important medical applications as a drug delivery system and food applications through the removal of cholesterol. These three polysaccharides are a demonstartion of the enormous range of structures and applications polysaccharides can achieve.

What evolution hasn’t achieve, well that’s where the rest of our project is taking us…

The aim of this mini project will be to create a three gene inducible plasmid. Each gene on the plasmid will 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 will allow the creation of a monosaccharide, a disaccharide and a trisaccharide. While this in itself will not create a library of polysaccharides available from a singe E. coli, it will be a proof of concept and will sit alongside a Tinkercell model showing 50 genes in a plasmid. This is important. Imagine being able to control the output of 50 genes or even 250 genes on a plasmid, each transcribing a different glycosyltransferase. The result would be a small polysaccharide factory without having to create a different model every time a new bespoke polysaccharide needs to be synthesised.

The project will be constructed using the Biobrick 3A assembly method. The product will be tested and then analysed by mass spectrometry to show the concept works.

My section of the project is construction of the three glycosyltransferase operons. This is going to be done through two different assembly methods with identical aims first by biobrick construction and then by the Gibson assembly method. The purpose if this is for a comparison of the assembly methods intended for future use by iGEM teams. Construction of a complete operon by either or both methods will ideally yield the predicted three polysaccharides, synthesised by the chosen sequence of glycosltransferases and the wzy-dependent system. The three operon variants with similar but not identical glycosyltransferase genes demonstrate the ability to construct different monosaccharide combinations within the repeat unit, an alternative approach to the three gene inducible operon project, undertaken by Freddie. The structure of the polysaccharides can be analysed by mass spectrometry. If the glycosyltransferases chosen do not work as hoped and time and funding allow, there is potential to order different glycosyltransferases and re-attempt the operon. Alex's work will hopefully confirm their efficacy in advance. We would hope to send the final operons as new parts to the parts registry for future iGEM teams to use as a biobrick.

So why do we need a database and what should it be capable of?

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

The database serves to communicate the work done in the lab with the scientific community in the sense that the user will be able input the repeating unit of their choice/design, and the database will return a list of enzymes/inducer compounds, necessary in order to produce said repeating unit.

This will improve the efficiency of the polysaccharide lab work and speed up the process of polysaccharide production.

It is not unforeseeable that there may be more than one enzymatic approach to producing a particular polysaccharide; as a long term aim of the database project, it may be possible that our database could return some of the advantages/disadvantages of certain construction pathways. Indeed if some repeating units are not possible, the database could suggest similar or alternative products with ‘nearly’ identical properties.

Combining this database with polysaccharide properties it could be possible to define the sugars and enzymes required based on the desired properties of the interface user.