Part:BBa_M36584:Experience
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Applications of BBa_M36584
Part BBa_M36587 was created with the goal of mimicking the acetoacetate production step of the Clostridium acetobutylicum acetone production pathway in E.coli. Since acetone cannot be used as a biofuel, its production is usually minimized or suppressed in cell or bioreactor engineering experiments with C. acetobutylicum. We, however, hoped to make use of this production of acetone for the degradation of polystyrene. Polystyrene (molecular formula [−CH(C6H5)CH2−]n), commonly produced as Styrofoam (expanded polystyrene foam), is classified as a durable, non-biodegradable plastic, yet it is often used in products designed for a short or one-time use due its low manufacturing costs. Unfortunately, this has led to a rapidly-increasing accumulation of polystyrene waste in the environment. Although styrene monomers and oligomers are susceptible to biodegradation, polystyrene is generally considered non-biodegradable as a result of its high molecular weight and highly-stable structure. Acetone has long been known to swell and depolymerize polystyrene, leaving behind a viscous liquid of styrene monomers and oligomers as the Styrofoam breaks down. This viscous styrene can then be reused as styrene glue, commonly used in industrial, craft, and construction applications. The ctfAB DNA was ordered from DNA 2.0 and transformed into 5-alpha commercial competent E.coli, after which liquid cultures were made.
We had planned to test for acetone production using two assays: 1. Test for the presence of acetoacetate production with the use of Bayer Ketostix (nitroprusside). 2. Perform a protein gel analysis to test for significant production of desired ctfAB proteins
1) To test for acetoacetate production, we needed to add acetic acid, the precursor/intermediate produced by the reaction before ctfAB in the natural C. acetobutylicum acetone pathway. Equal amounts of ctfAB liquid culture at OD 0.5 were added to 14mL round bottom tubes. To each tube, increasing concentrations of acetic acid were added in order to determine not only if the cells were producing acetoacetate, but also at what concentration of acetic acid they produced the most. six tubes were created, containing:
ctfAb cells ctfAB cells + 0.1% acetic acid ctfAB cells + 0.5% acetic acid ctfAB cells + 1% acetic acid ctfAB cells + 5% acetic acid ctfAB cells + 10% acetic acid
These were tested for acetoacetate production every 30 minutes for 1.5 hours using Bayer Ketostix. Bayer Ketostix are commonly used to test for acetoacetate (acetoacetic acid) in the presence of urine, a common sign of ketoacidosis in diabetic patients. The Ketostix change from a pale nude color to dark red in the presence of acetoacetate, but unfortunately all of our tests came back negative, indicating that there was no production of acetoacetate in the ctfAB cells at any concentration of acetic acid.
One possible explanation for the negative results is that acetoacetate readily undergoes a hydrolysis reaction that splits the molecule into acetone, which is not picked up by the Ketostix. This also created further problems in our later experiments testing for the prodution of acetone. 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. We were also unsure if the acetic acid was killing the E.coli, or if minute amounts of acetoacetate produced could be killing the rest of the cells in the tube.
2)Given the above challenges, we decided to run a protein gel to test if our part was producing ctfAB proteins at all. The size of the ctfA and ctfB proteins produced by the ctfAB gene are 26.3 kDa each, so would look for significant bands at the corresponding location on the gel. We added equal amounts of liquid culture at OD 0.5 to four different 1.5mL tubes and induced protein expression in 0, 10, 100, and 1000mM concentration rhamnose respectively. This was done so that we would see a band in the same location of the gel increasing in thickness across the gel as the concentration of rhamnose increased. Please click the protein gel image for significance and location of columns of interest.
Our results showed that it was plausible that ctfAB proteins were being produced. The leftmost columns show the ladder, ctfAB + 1000mM rhamnose, ctfAB + 100mM rhamnose, ctfAB + 10mM rhamnose, and ctfAB + 0mM rhamnose in order from left to right. Since our proteins are predicted to be around 26.3 kDa, they should appear between the third and fourth bands of the ladder. A significant band increasing in thickness along with increasing rhamnose concentration right to left is seen on the gel, but it is slightly above the fourth band, out of the predicted range. This is most likely our protein, its elevated position possibly being due to an abundance of positively charged amino acids affecting its travel through the gel. It could due to many possible errors in the protein gel process, but the increasing thickness with rhamnose induction makes it plausible that it is showing our ctfAB proteins.
User Reviews
Transformation of the ctfAB part into E.coli only worked with the addition of SOC outgrowth media to plates, and liquid cultures were found to need SOC outgrowth media as well.
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