Team:UIUC-Illinois/Project/Future/Petrobrick

From 2012.igem.org

As a side project, we decided to characterize a previous team’s work on an existing biobrick. For that purpose, we chose to characterize the University of Washington’s Petrobrick. The Petrobrick, once transformed into E. coli, acts as a microbial alkane production pathway. Two enzymes are co-transformed to create this biobrick: Acyl-ACP Reductase (AAR - Bba_K90032) and Aldehyde De-Carbonylase (ADC - Bba_K90031).

AAR reduces cellular fatty acyl-ACP from bacterial fatty acid via into fatty aldehydes. ADC then removes the carbonyl group on the fatty aldehyde, resulting in an odd number alkane chain one carbon shorter than the original Acyl-ACP fatty acid. In turn, both of the enzymes convert fatty acids into an odd number alkane by means of a constitutive protein expression plasmid.

Click here to read our submission on the Parts Registry experience page for the characterization of the Petrobrick.

It was noted that the alkane production is enhanced when growing expression strains using the optimized growth conditions developed by the 2011 University of Washington team, so we followed the protocol to the best of our ability. After analyzing their results, we decided to reproduce the experiment specifically to test for the production of C15 alkanes, which were the most abundant. In order to do so, four samples of empty E. coli cells grown in TB media were injected with known concentrations of C15 alkanes (obtained from Sigma-Aldrich).

They were used as control samples with the corresponding concentrations: 1 mg/L, 10 mg/L, 50 mg/mL, and 100 mg/L. After injecting the cells with the known C15 alkanes, the cells were then incubated for 48 hours in M9-Glucose media to ensure nothing else changed in the development of the control samples. After incubation, Ethyl Acetate was used to extract 200 uL of the alkane samples to be analyzed with GCMS. Gas-Chromotography Mass-Spectometry (GCMS) was used to create a standard curve of the four known concentration and their corresponding peak areas. For the actual samples, the Petrobrick-transformed dh5a E. coli cells were grown in TB overnight. After growth, the cells were spun down and re-suspended in M9-Glucose media for 48 hours. Ethyl Acetate was used to extract the produced alkanes. 200 uL of each of the samples were used for GCMS analysis.

Fig. 1. Standard curve created from the results of GCMS analysis of the four controlled known concentrations of C15 alkanes and the corresponding peak areas.

Fig. 2. Concentration yields of C15 alkanes from the four experimental samples based on standard curve measurements at corresponding retention time for pentadecanoic acid (C15 alkane)

The average yield for C15 alkanes as determined by our results was 160.2 mg/L. Our maximum yield was 190.6 mg/L. The average C15 alkane yield for the UW team was 160.3 mg/L. Based on our results, we were able to successfully reproduce the results from the UW iGEM team’s work on the Petrobrick, effectively proving its function.