Part:BBa_K590025:Experience

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User Reviews

UNIQ3c2e4c6d6f4086df-partinfo-00000000-QINU

••••• 2012 UIUC iGEM

As a side project, we decided to characterize the University of Washington's existing biobrick, the Petrobrick. After analyzing the results from their experiment, we decided to reproduce the results specifically for the detection of C15 alkanes, as they were the most abundant. Transformed cells were grown in TB media and then subsequently in M9 min-glucose media to produce the alkanes. Using Gas Chromotography-Mass Spectometry, a standard curve was created using the known concentrations of four control samples of C15 alkanes and their corresponding peak areas. Then, GCMS analysis of four samples was used to find the concentration yields based on the standard curve. The average retention time for C15 alkanes was determined to be at 11.47 s, according to the chromatogram analysis. Our data was in sync with the University of Washington results, as their average yield of C15 alkanes was 160.3 mg/L and our range was from 130 to 190 mg/L. Based on our results, we were able to confirm the function of the Petrobrick.

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••••• 2012 UCalgary iGEM

The 2011 Washington iGEM team developed the PetroBrick, a BioBrick consisting of two primary genes. These include acyl-ACP reductase (AAR), which reduces fatty acids bound to ACP to fatty aldehydes, and a second gene called aldehyde decarbonylase (ADC), which subsequently cleaves the entire aldehyde group and results in a hydrocarbon chain. Essentially this allows for hydrocarbons to be produced from glucose. What we realized though, is that the fatty acids that the PetroBrick targets, have a very similar structure to NAs. This lead us to believe that the PetroBrick may have the potential to turn NAs in to hydrocarbons and be a perfect solution to remediating NAs! First though, we needed to show that the PetroBrick did in fact work as expected. We had some difficulty with the DNA from the registry and had to request the constructs directly from the Washington team. Once we had the Petrobrick, we needed to verify that the Petrobrick would work in our hands as it did for the 2011 Washington team. Figures 2 and 3 demonstrate the function of the Petrobrick. Figure 2: Gas Chromatograph demonstrating the differences in peak composition between an E.coli control and the Petrobrick. There was a large increase in a peak with a retention time of 12.25 min. suggesting that the Petrobrick was producing a new compound. Figure 3: Mass Spectra of the gas chromatograph peak at 12.25 min. The spectra suggests that the Petrobrick is selectively producing a C15 alkane. This is what was expected as determined by the Washington 2011 iGEM team. With the Petrobrick shown to be able to successfully produce alkanes, it was time to test it out on NAs, to see if they could be selectively converted into alkanes! This experiment used commercially available NAs fractions including a large number of different complex NAs compounds. Figure 4: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. NAs were used as a substrate. A NA standard was required to compare peaks. Figure 5: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 2, using NAs as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared. The above graphs indicate that hydrocarbons were successfully produced from E.coli that contained the PetroBrick plasmid, as analysed with GC-MS. In Figure 2, E.coli containing the PetroBrick had significantly higher hydrocarbon peaks than in a control of E.coli that did not contain the PetroBrick plasmid. Not only was the PetroBrick able to degrade NAs into alkanes, but it was also able to produce alkenes as shown by Figure 3, indicating that the PetroBrick worked how we had expected it to! Special thanks to the Washington 2011 iGEM team for sending us their PetroBrick plasmid stock as the one from the registry was non-functional.

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••••• 2016 USP-EEL-Brazil

Since we would be using a part of this composite in our own project, we decided to prove that the Petrobrick was really working for us. To do that, we transformed E.coli cells with the PetroBrick composite and amplified the part with them, followed by a miniprep. Then, using the purified plasmids, we transformed another batch of E.coli and inoculated these ones into optimum medium. Following up the cell's growth, we then had to analyze them to check whether it was really working or not. The chosen method for analysis ended up being the Gas Chromatography, in which we used a 5% polar column. The cell suspension was splitless injected with N2 at 250ºC. After that, the initial temperature was kept at 120ºC for 0,5min and increased to 280ºC at a 3ºC/min rate. The sample was then incubated for 6.17min. Unfortunately, we had no way to make a standard curve and no access to a mass spectrophotometer whatsoever. Gas chromatography results. Source:USP-EEL-Brazil. Due to our limitations, we can't give a bulletproof grantee, but we strongly believe that some of these peaks are the desired alkanes we were looking for, once all the substances that came out are definitely long-chained carbons. Our lack of time resulted in the analysis official inconclusiveness.Therefore, we agreed between ourselves that it worked fine for us and we would go on to use their part in our project.

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