"

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, BBa_K1423006, contains a heavy metal iducible promoter (BBa_J33201),and a construct consisting of a ribosome binding site, the ATF1 gene, and a double terminator (BBa_J45199).The second biobrick, BBa_K1423007, consists of a high strength constitutive promoter (BBa_J23100), a ribosome binding site, the ATF1 gene, and the double terminator (BBa_J45199).

BBa_K1243007: Inducible Banana Odor Generator

BBa_K1243008: Constitutive Banana Odor Generator

Quantification of ATF1 through Gas Chromatography and Mass Spectroscopy

Before we could quantify our results through gas chromatography, we had to make sure that the liquid LB agar that cells were grown would not interfere with our ability to read the presence of isoamyl alcohol and isoamyl acetate. Included below is a chromatograph showing both of these compounds injected into LB form cell growth until at 5mM concentration. To ensure they were correct, we simultaneously performed a mass spectroscopy of this sample, whose results verified that these compounds were correct, as their molecular makeups were analogous to isoamyl alcohol and isoamyl acetate.

LB solution injected with 5mM isoamyl alcohol and 5mM isoamyl acetate.

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 cell’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. In the last procedure, we performed 10 different trials 3 times each. The constitutive promoter was used as a theoretical maximum possible yield. from the isoamyl acetate output observed, we used it as the base standard that the other results were based upon. The yields were based upon the Corrected abundance are of the peaks identified through mass spectroscopy as isoamyl acetate. The results of the other trials and their errors were normalized to the value of the constitutive promoter.

Normalized results of the readings of isoamyl acetate presence.

From these graphed results (not including all of our negative controls, see protocol) we have obtained a variety of different results, despite the large variances from the standard error readings.

As expected, the constitutive promoter without isoamyl alcohol did not produce isoamyl acetate.

Conversely, when provided isoamyl alcohol, the acetate is produced, which is used for our expected maximum output that all other results have been normalized to.

Unexpectedly, when provided isoamyl alcohol but no arsenite, the arsenic induced promoter still produced isoamyl acetate, at what may be a slightly larger rate. Due to our error, we can’t confirm this behavior, but it is apparent that the arsenic induced repressor only weakly binds to the promoter site. Which suggests that this promoter may not be suitable for certain tests that rely specifically on the presence of arsenic or other heavy metals

Over the next five columns, we notice a very specific downwards trend. It appears that, although arsenic must bind to the repressor for heightened functionality, the increasing concentrations of arsenite may be toxic for cells. After 0.5uM of arsenite, the amount of isoamyl acetate produced drops significantly, suggesting that cells are dying off, so that less isoamyl acetate is produced.