"

From 2010.igem.org

Contents 1 Hydrocarbon Solubility 1.1 Aim 1.2 Proposed Method 1.3 Step 1: AlnA BioBrick Formation 1.4 Step 2: OprG BioBrick Formation 1.5 Step 3: Characterization - Emulsifying activity

Hydrocarbon Solubility

Aim

To create BioBricks for the production of emulsifiers AlnA and OprG. These biobricks will be implemented in Escherichia coli K12 and characterized and evaluated on their emulsifying capabilities.

Proposed Method

In order to overcome the mass-transfer limitations of an oil-water environment, the production of an emulsifier is proposed. The presence of an emulsifier will increase the amount of alkanes dissolved in the water phase, increasing the availably for the microorganisms. Two emulsifiers from naturally oil-degrading bacteria will be expressed in E.coli K12 using the BioBrick method.

Step 1: AlnA BioBrick Formation

Based on: Gil Walzer, Eugene Rosenberg and Eliora Z. Ron. The Acinetobacter outer membrane protein A (OmpA) is a secreted emulsifier. Environmental Microbiology 8(6): 1026–1032 (2006)

Oil-degrading strains of Acinetobacter produce a variety of high-molecular-weight biosurfactants. One of the best-studied emulsifiers is Alasan produced by Acinetobacter radioresistens (KA53). It contains three proteins of 15 kDa, 31 kDa and 45 kDa. The 45 kDa protein is associated with the major emulsifying ability and is called AlnA. AlnA is a homologue of the E. coli OmpA.

The necessary gene, AlnA, will be synthesized, and the appropriate RBS and promoter that will be used are existing BioBricks. The selection criterion for RBS and promoter for each construction will depend on the results obtained during the RBS characterization experiments.

Step 2: OprG BioBrick Formation

Based on: Gil Walzer, Eugene Rosenberg, Eliora Z. Ron. Identification of outer membrane proteins with emulsifying activity by prediction of β-barrel regions. Journal of Microbiological Methods 76:52–57 (2009)

OprG is another emulsifier, an outer membrane protein originating from Pseudomonas putida KT2440. The necessary gene, OprG, will be synthesized, and the appropriate RBS and promoter that will be used are existing BioBricks. The transformed cells will be cultivated in shake-flasks of 250ml.

Step 3: Characterization - Emulsifying activity

Strains:

• AlnA: E.coli K12/200C
• OprG: E.coli K12/201C
• Negative control: E.coli K12
• Cultivation: 250 ml shake-flasks / solid agar plates

In order to determine whether the emulsifiers are produced, if they are excreted or not, and how well they emulsify, a characterization procedure will be performed. This will consist of the production of the emulsifier (to be shown using SDS PAGE), and an emulsification test with cell-free supernatant, purified cell extract and purified whole cells (washed). The emulsification test will occur by creating an alkane/water bi-phase mixture and determining the absorbance before and after addition of the emulsifier, and/or by shaking the sample vigorously and measuring the height of both phases before and after.