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. Promoter strengths will be quantitatively determined from the new promoter+RBS sequences we will be adding to the registry.

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 [1-2]. 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.

[1] Lundborg, M., Modhuker, V. & Widmalm, G. (2010) Glycosyltransferase functions of E.coli O-antigens, Glycobiology. 20:3, pp. 366-368.

[2] Woodward, R. et al. (2010) In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz, Nature Chemical Biology. 6, pp. 418-423.

Synthesis of polysaccharides provides us with an endless list of applications in medicine, industry, cosmetics and engineering that we are able to demonstrate. Expression of Levansucrase from Bacillis subtilis into our E.coli, targeting the enzyme to outside the cell, to produce levan, a polymer of fructose. Levan is a potential glue, used by Newcastle Igem 2010 (BacillaFilla) to increase the lifespan of man-made structures. Levan is also a functional biopolymer in foods, feeds, cosmetics, and the pharmaceutical and chemical industries. We are separately expressing hyaluranon synthase in E coli to produce the glycosoaminoglycan, hyaluranon. Hyaluranon is an important component of the human cellular matrix and has medical applications in surgery and the cosmetic industry, especially skin care. Finally we are producing cyclodextrin, a cyclic oligosaccharide to exhibit the alternative structures that can be produced in E coli other than fibres. Cyclodextrins have pharmaceutical and food industry applications. These examples are just a few of the diverse potential that synthetic biology can achieve.

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.