Part:BBa_M36553:Experience

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Applications of BBa_M36553

Can potentially be used for the reduction of citral into citronellal (which is a key component of citronella oil, an insect repellent). If citronellal is made effectively, a citronellal-producing device can be engineered and distributed around the world to reduce the mortality rate due to insect-borne diseases.

Our experience, however, was not able to successfully reproduce the above function, so please see the performance data in the following section for more details/caveats.

User Reviews

UNIQb4653400e6a82f33-partinfo-00000000-QINU UNIQb4653400e6a82f33-partinfo-00000001-QINU

Initial Transformation and Antibiotic Selection Plate Analysis: After allowing our plates to grow overnight, there were bacteria colonies present in our LB+Kan plates and no bacterial colonies in our LB+Amp plates. This indicates that our plasmids, which contained a Kan resistance gene, were successfully transformed in our E. coli bacteria.

OD600 Measurements: Before performing a Western blot, we measured the OD600 of each of our samples to ensure that a sufficient concentration of cells was present in each of our clonal stocks. Each of the OD600 values was above 1.400, which we deemed sufficient to start preparing our Western blot (which was done by diluting each sample to 0.4 OD600).

Western Blot Analysis The size of OYE6 is 41.2 kDa. As such, we used 15% gel and expected to see bands around the 5th and 6th band markers on the ladder. The negative control, with p70417 plasmid, showed an unusual pattern that is not typical of OYE6 presence. For the samples with OYE6, the Western blot membrane had a very light blue band between the 5th and 6th ladder bands for each of our samples with our designed plasmids (in concurrence with our expectations). These results indicate that our enzymes were successfully translated into desired OYE6 proteins in the bacterial cells, albeit in small amounts.

Functional Assay Analysis: After 2% or 4% citral was added to the prepared samples, the samples were fairly clear with a strong lemon scent. After one day of storage, approximately half of our frozen supernatants were clear, while the others were yellow, with a very yellow, oily substance at the top of the frozen media. After a full week of storage at -20 degrees Celsius, all of the supernatants were very orange in color, with dark orange specks visible throughout. We suspect that this change in color is due to the natural characteristics of citral (and possibly citronellal, if produced) rather than any unexpected reactions within the cells. The supernatants smelled strongly of lemon candy. Since citral and citronellal have a similar smell, we cannot determine if citronellal was produced based solely on smell (our p70417-plasmid-containing negative control also had a lemon scent due to the presence of citral). The cell pellets were quite orange as well. This could indicate that citral and possible citral products were still in the bacterial cells after spinning the sample down. Since we did not test our cell pellet for the presence of citral/citronellal, it is possible that our results underrepresent the citral remaining in the cells and the citronellal produced in the cells.

GC-MS Results: Based on the testing of our standards, citral has a retention time of approximately 8.5 minutes. The major peak in the mass spectrum was at 69 m/z. From online resources, we found that citronellal should have MS with its highest peak at 41 m/z, second highest peak at 69 m/z, and third highest peak at 55 m/z. However, we did not get any results that had an MS with a peak at 41 m/z; in fact, most of our results had MS with peaks higher than 55 m/z. As such, we were not able to obtain any information that would confirm the existence of citronellal in our cells. Because we did not have our own GC-MS data for citronellal, we made our conclusions primarily based on the amount of citral present in each of our samples. We did this by comparing the percent area of the MS peak at 8.5 minutes (which reflects the amount of citral in the solution) to those of our negative controls. If our enzymes functioned properly, the percentage of citral in the sample would be lower in the samples that contained our plasmid. We found that this was generally not the case; the percent area of the peak of the samples containing our plasmid was close to (and usually greater than) the average percent area of the peak of our negative controls (i.e., the bacteria containing the p70417 plasmid). We also observed another high peak in our GC at 8 minutes that does not contain the 41 m/z peak but does contain the 69 m/z and 55 m/z peaks, which is similar to the MS for citral and citronellal. This unique result makes it unclear as to whether citronellal was actually formed in the cells; this peak could indicate the presence of some other citral product, but its exact identity is unclear. As a whole, the results of GC-MS were inconclusive. From our data and based on our preparation procedures, we observed that OYE6 is not an effective enzyme for the reduction of the alpha-beta unsaturated double carbon bond in citral. At the same time, we recognize that our experiments may have suffered from sources of error. There are follow-up experiments that we can perform to better understand the mechanisms of OYE6, which could still potentially be used to reduce citral to citronellal.