- Assembled and sequence verified Venus cassette plasmid and pp2551 cassette plasmid
- Assembled the multigene sensing assembly
- Tested another version of the sensing assembly and demonstrated that it works
Our team did not have an assay to get quantitative measurements concerning the production of L-DOPA in our production assembly. However, by itself, L-DOPA will oxidize to melanin. As a result, to get qualitative results concerning our production assembly, we grew up our production cells in LB media containing 5 mM tyrosine and 0 mM tyrosine and then observed if a color change occurred. The diagram above shows such a color change. Our production assembly did observe a similar color change, but the change in color was not very pronounced.Sensing Circuit
We tested a sensing construct that was similar to our sensing construct in design but built with different methods and parts. Instead of Venus fluorescence protein, this sensing circuit expressed e2-crimson, and was inserted into a pMMB67EH vector, while in our sensing construct, the Venus gene was inserted into a pET28 vector. Both constructs were transformed into Top 10 E. Coli strain and used in the fluorescence detection assay. We found that the fluorescence intensity in the cell increased as the amount of L-DOPA in the media increased for the lab construct. However, we found that our sensing construct, in the presence of L-DOPA, showed increased expression of fluorescence when certain amounts of L-DOPA were present in the media. You can see a slight increase in fluorescence from no L-DOPA to 100 uM of L-DOPA. However, the data shows that a lower level of fluorescence was detected when 1 mM of L-DOPA was present, which is different than the predicted outcome. These data suggest that different combination of vector plasmid and E. coli strain might have a different effect on the inducibility of sensing construct. Therefore, further study is needed to find out the best combination of vector and E.coli strain for this sensing construct.
Fig. 1. A chart showing the detected level of fluorescence (E2-Crimson) expressed by a sensing cell in different conditions of L-DOPA in the media.
Fig. 2. A chart showing the detected level of fluorescence (Venus) expressed by a sensing cell in different conditions of L-DOPA in the media.