"

From 2010.igem.org

Contents 1 Overall project 2 Project Details 2.1 The Experiments 2.2 Part 3: Results

Overall project

Standardization of the Rhamnosiltransferase 1 gene complex (rhlAB) into a Biobrick-friendly for rhamnolipid production in E. coli.

There is considerable interest among bio-industries in bioremediation products such as Rhamnolipids. Rhamnolipids as biosurfactants are important in the remediation of oil spill areas. The cleanup of the Exxon Valdez oil spill with rhamnolipids as biosurfactants was too expensive and complicated, therefore impractical for large-scale bioremediation. However, advances in genetic engineering and synthetic biology offer a viable solution to oil spill pollution clean up. In this project we use genetic engineering as a tool to integrate genetic parts by the biobrick assembly standard protocol of iGEM to develop a biobrick-friendly for rhamnosiltransferase 1 complex (rhlAB) gene expression in Escherichia coli for standardized rhamnolipid production. Our biobrick integrates a promoter, a RBS (ribosomal binding site), our gene sequence of rh1AB isolated from Pseudomonas aeruginosa, a GFP reporter and a terminator. All the parts fit into a plasmid backbone that can be transformed into E. coli strains, which can then produce rhamnolipids.

Project Details

The Experiments

The purpose behind our project is to take a gene that produces the Rhamnosyltransferase 1 enzyme in a pathogenic bactria known as Pseudomona aeruginosa and insert it into an efficient and not so pathogenic bacteria that is Escherichia coli. This will help create a rhamnolipid that can aid in the mosre efficent degradation of hydricarbons.

We'll start out by selecting that components of our gene which will be inserted into the E. coli for expresion. We have chosen a promoter, a RBS, a reporter and a terminator. All of this from the plates that our friends at iGEM have conviniently sent all of us. These plasmids will then be used to obtained transformed bacteria that have those parts in them. We'll have to choose which colonies are the ones that have our genes in them; and the colonies themselves will help us do this. The colonies that have been successfully transformed will be white and the ones that haven't, will be blue. Now, we can't use the entire plasmid for our construction. We have to extract that gene for the promoter, RBS, reporter and terminator from the plasmid to be able to use them, so we'll perform minipreparations to be able to achieve that.

In the meantime, we have to obtain our gene from the P. aeruginosa. This bacteria is donated to us by Marcelino Gutierrez, Ph.D, who is a researcher from the chemistry department at INDICASAT-AIP. We'll take some colonies and grow them in liquid media and then we'll extract the genomic DNA. Our DNA of interest wil be yielded by performing a PCR.

Once we have this, we have to mutate the Rhamnosyltransferase 1 gene to eliminate the restriction sites that clash with the iGEM disposition that indicates that our gene cannot be cut in any way by the restriction enzymes.

Part 3: Results