Team:Uppsala/Biosurfactants
Biosurfactants
The aim of our group was to make a synthetic system in Escherichia coli expressing mono-rhamnolipids. Rhamnolipids are biosurfactants that can be used for improving bioavailability of PAHs. (Chen et al. 2013). PAHs, that we attempt to break down, tend to cluster due to their hydrophobic properties. The bioavailability of PAHs increases in the presence of rhamnolipids, improving their degradation by the enzymes laccase and dioxygenase. 3A assembly was used to assemble the genes rhlA and rhlB responsible for the mono-rhamnolipid production together with RBSs and a promoter. Assembly steps were verified by Sanger-sequencing. Different assays were used to verify the production of rhamnolipids in our system, among others drop collapse test, CTAB test and thin layer chromatography. The results indicated the presence of rhamnolipids.
Introduction
Breaking down PAHs extracellularly is problematic since they aggregate into clusters due to their hydrophobic properties and the efficiency of the enzymes laccase and dioxygenase will drop dramatically due to this. To counteract these cluster formations our strain of Escherichia coli will produce biosurfactants, namely mono-rhamnolipids, figure 1. This biosurfactant is made up of a hydrophilic rhamnose head and a 3-(hydroxyalkanoyloxy) alkanoic acid (HAA) fatty acid tail which can vary in length (Han et al. 2014). The mono-rhamnolipid will interact with and disperse the aggregated PAHs, increasing the efficiency of the enzymes that eventually will break down the PAHs.
Using biosurfactants to disperse hydrophobic compounds is nothing new. Many microorganisms use biosurfactants to access carbon that would otherwise be unavailable to them. The bacteria Pseudomonas aeruginosa is one of the best characterized organisms that produces rhamnolipids (Oschner et al. 1995). Besides mono-rhamnolipids they also produce di-rhamnolipids which has two hydrophilic rhamnose heads (Moussa et al. 2014). These are not as good at dispersing hydrophilic compounds and will not be expressed in this project.
The enzymes responsible for producing the mono-rhamnolipids are called RhlA and RhlB. These make up the subunits of rhamnosyltransferase I and are organized in an operon for highest efficiency. The RhlA creates the fatty acid tail and the RhlB connects the tail to a rhamnose molecule. (Han et al. 2014) The genes that express these enzymes are labeled rhlA and rhlB and both of these genes were already present in the iGEM distribution kit. To express the genes a promoter was added together with RBSs at the sites needed, all added using 3A assembly.
To detect the mono-rhamnolipids a few assays were used. Drop-collapsing tests were used to confirm the presence of biosurfactants and CTAB plating were used to confirm that rhamnolipids were produced. Finally to confirm them as mono-rhamnolipids a TLC and mass spectrometry was conducted.
System design
For our project we wanted a system that only expressed mono-rhamnolipids, see gene construct, figure 2. Biobricks (BBa_K1331001) (rhlA) and (BBa_K1331004) (rhlB) encode for enzymes Rhl A and Rhl B which together makes up rhamnosyltransferase I and are responsible for biosynthesis of mono-rhamnolipids. A constitutive Anderson promoter was used for the system to always express the genes.
In the original operon rhlA and rhlB are expressed together with rhlC, which encodes for rhamnosyltransferase II (Rahim et al 2001) and regulatory genes rhlI and rhlR (Pearson et al. 1997). Since only mono-rhamnolipids are desired in our construct, rhlC is not incorporated into our construct. Expression of rhlA and rhlB is possible without the regulatory genes and they were as well not included in the final construct.
Method
Assembly
3A assembly was used to assemble the parts that were required to build the construct from the iGEM distribution kit. First RBS, (BBa_B0034) was assembled with gene rhlA and rhlB respectively into (BBa_K1688002) and (BBa_K1688003). This was followed by assembly of BBa_K1688002, BBa_B0034 and BBa_K1688003 into BBa_K1688001. An Anderson promoter (BBa_J23101) was assembled with this part to give the final construct, (BBa_K1688000). The length of the assembled part was confirmed using gel electrophoresis at each stage and correct assembly was verified using Sanger sequencing.
For assembly steps E.coli, strain DH5α was used as a chassi since it gives good plasmid preparation results and high transformation frequencies. (Forster et al. 2014) The final construct was transformed into E.coli, strain BL21DE3 since it gives better protein expression.
To verify expression of genes rhlA and rhlB the final construct was assembled together with (BBa_K1688004), a dTomato construct consisting of an RBS and a gene encoding for a modified RFP. This gives the bacteria a unique red fluorescent colour when the genes are expressed.
Characterization
A couple of assays were performed to determine the presence of mono-rhamnolipids. First of all a drop collapse test was conducted for confirmatory reasons. In the presence of biosurfactants, the drop collapses due to the decrease of surface tension of the hydrophobic surface (oil). In the absence, the drop does not collapse and remains intact like a bubble. (Walter et al. 2010)
To further determine the presence of mono-rhamnolipids CTAB (cetyl-trimethyl-ammonium bromide) plating was used. This is a widely used method for detecting rhamnolipids. Agar plates containing the CTAB were made using a standardized protocol. The plates contain methylene blue and CTAB. The rhamnolipids forms a complex with the CTAB and forms a halo around the colony. One can thus conclude that if there is a halo, rhamnolipids should be present. (Siegmund and Wagner. 1991)
Both the drop collapse test and the CTAB test are nonspecific and only show indication that there are biosurfactants and rhamnolipids respectively present. To confirm the presence of mono-rhamnolipids, a thin layer chromatography (TLC) was conducted of the sample and with standard mono-rhamnolipids as reference. Mono-rhamnolipids were extracted from bacterial supernatant by solvent-extraction method using ethyl acetate and run on TLC silica plates using orcinol-sulphuric acid as a staining solution (Laabei et al. 2014). The extracted lipids were also sent for mass spectrometry analysis for further confirmation.
To view our results from this part of the project, please click here.
References
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