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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 biosynthezise a rhamnolipid that can aid in the more efficent degradation of hydrocarbons.

We'll start out by selecting the parts or components of our BioBrick which will be inserted into the E. coli for expresion of rhamnosyltranferase gene enzyme for rhamnolipid production. We have chosen a promoter, a RBS, a reporter and a terminator. All of the parts came from the collection of genetic parts that our friends at iGEM have conviniently sent all of us as a kit. Our chosen parts are well characterized and were been tested that works efficiently with the assembly standar protocol. These plasmids will then be used to obtain transformed bacteria that have those parts in them. 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. They were given to us in liquid media ready for extraction of genomic DNA. Our DNA of interest will be yielded by performing a PCR.

Once we have this, we have to mutate the Rhamnosyltransferase 1 gene to eliminate the restriction site of Pst1 that clashes with the iGEM disposition for design and BioBrick construction that indicates that our gene cannot be cut in any way by the restriction enzymes of the assembly standard protocol. This mutation will allow us to have the same gene without the restriction sites within our construct permitted. To resolve this issue we propose to do a multiple site mutagenesis.

In the end, we'll be able to ligate all the parts together to have one general section that contains the Promoter+RBS+ OUR GENE +Reporter+Terminator and EUREKA we bring a new BioBrick ready to be characterized.

Part 3: Results

We were able to standardize the biobrick extraction procedures and assembly of the parts once they were ready. Every time each of the parts had been cloned in the bacteria, we performed gel electrophoresis just to check if the transformation of the bacteria had taken place. We took colonies from each part and performed minipreparations and subsequent digestions. We then prepared an agarose gel and were able to see each of the parts. EUREKA!!! Once each of the parts were obtained, we were able to ligate them together (promoter + RBS and reporter + terminator) and this was confirmed by performing several gel electrophoresis.

We also were able to introduce the cloned Rh1ab gene into the pGEMT Easy Vector.

Mutagenesis procedures