Part:BBa_M36587:Experience

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Applications of BBa_M36587

Part BBa_M36587 was created with the goal of producing acetone from a simple starting material, acetoacetic acid. The application of this part could have monumental positive effects on environment, as it would be able to "melt down" Styrofoam. Styrofoam is a waste product in many industrial pathways and does not degrade in the environment for several years. By using acetone on the Styrofoam, it would take up less space in land fills and could be reconverted into another product (potential glue) for further industrial use.

We had planned to test for acetone production using two assays:

1. One assay was to use polystyrene as an indicator for acetone production. Acetone quickly converts polystyrene to a sticky, glue-like material, that is devoid of the air pockets it once held onto.

2. A second assay was to use ketostix (nitroprusside) to test for the presence of acetoacetic acid. The goal was to see less acetoacetic acid over time, as it would hopefully be converted to acetone.

However, we quickly found that our starting material, acetoacetic acid, posed many problems during experimental assays. Using a hydrolysis reaction on ethyl acetate, acetoacetic acid could theoretically be made. When we tried to produce the starting acid though, we found that it was nearly impossible to make. This is because in the middle of acetoacetic acid formation, a chemical reaction occurs which splits the intermediate into an acetone molecule and another side product. Acetoacetic acid is incredibly unstable and therefore is very hard to obtain; even if this chemical was to be synthesized, its acid form only has a half life of 140 minutes in room temperature water.

Because of this chemical synthesis problem, we had no way to properly assay for the production of acetone. Even if we had the chemical, it would degrade to acetone slowly every time, which would pollute our assay for acetone in the first place. The degradation also would conflict with testing for the presence of the chemical, as we would have no way to differentiate whether the chemical was being converted to acetone by the part, or simple degrading to acetone over time.

Given the above challenges, we decided to run a protein gel to test if our part was producing a protein. The size of the ADC protein produced by the ADC gene is 27.5 kDa. We grew bacterial pre-cultures to OD 0.5 and induced protein expression in 0, 10, 100, and 1000mM concentration rhamnose. Please click the protein gel image for significance and location of columns of interest.

As shown by the above protein gel, it is difficult to see a clear band between the 25 and 35kDa marks (close to 27.5kDa). Because the proteins in that general area get darker with increasing concentration of rhamnose, it is challenging to have a clear indication of whether or not this part produced unique ADC protein.

In conclusion, our assays were unable to test for the production of acetone, but the E.coli cells could have possibly produced a protein (still unclear) that came directly from this engineered part, BBa_M36587. Increasing protein concentration with increased concentration of rhamnose was our only successful data for this part during the 4 week experimentation cycle.

User Reviews

UNIQa1e4d6d31d28d7b3-partinfo-00000000-QINU UNIQa1e4d6d31d28d7b3-partinfo-00000001-QINU

This part was relatively easy to work with. However there was one exception.

when we received the DNA for this part from DNA synthesis company, DNA2.0, we had great difficulty transforming it into commercial chemically competent cells. We had to spread 1mL of 20% glucose on our LB-KAN plates to get growth of transformed cells. To give future advice for any group working with this part, please use glucose when executing transformations, growth periods, or any other experimental work.