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From 2014.igem.org

Better Biobrick Characterization

Characterization of the Efficiency of the ATF1 Enzyme

The ATF1 enzyme converts isoamyl alcohol to isoamyl acetate, which gives off a strong banana odor. The conversion of isoamyl alcohol to isoamyl acetate can be monitored using gas chromatography and mass spectroscopy. Thus, the efficiency of the ATF1 enzyme can be characterized.

We have constructed two different biobricks that include the ATF1 gene. The first biobrick, K1423007, is a construct consisting of an arsenic inducible promoter, a ribosome binding site, the ATF1 gene, and a double terminator. The second biobrick, K1423008, consists of a high strength constitutive promoter, a ribosome binding site, the ATF1 gene, and the double terminator.

BBa_K1243007: Inducible Banana Odor Generator

BBa_K1243008: Constitutive Banana Odor Generator

Quantification of ATF1 through Gas Chromatography and Mass Spectroscopy

In order to efficiently quantify the performance of these two constructs, we designed and performed a number of experiments based around E.Coli with the constitutive promoter plasmid, and E.Coli with the arsenic inducible promoter. The full protocol is located in our protocol section, and can be located through this link. Some findings through these experiments revealed that:

The arsenic based repressor may be affected by cadmium, leading us to believe that the construct can be affected by other heavy metals.

We based our concentrations off of MIT experience results on their BBa_J45200 experience page. But in our findings, the amount of isoamyl acetate is dwarfed by the 5mM concentration of isoamyl alcohol. The cells’s acetate output is very small compared to the initial alcohol injection. This leads us to believe that the production of isoamyl acetate can be misinterpreted based on odor tests alone due to its potency.

Although inducible by arsenic, cells appear to die off after the concentration of arsenite exceeds 5uM

Yields of isoamyl acetate were improved slightly when there was less open air within the incubation tubes

Our final Experimental procedure combined our best practices across our previous experiments.