Team:Imperial College London/Results

Results | Overlay of the two spectra from the cell cultures after catechol addition, reveals a broad peak appears with maximum at 380nm unique to XylE expressing cultures.

assay

Results | The threshold concentration of catechol needed for a person to identify a yellow positive test was found to be 30-40μM. This result was fed back to the modelling team and allowed them to constrain the concentration values of catechol to be used. Furthermore, it facilitated the determination of conditions for modelling of 2-step vs 1-step amplification. Two step was shown to be better. The prediction t for our system is that 2-step amplification output will be visible around 4 minutes earlier than the 1-step amplification system.

of C(2,3)O in whole cells

Results | Very useful information was acquired out of these assays of which the most important is the reaction profile of catechol(2,3)dioxygenase with catechol. The course of the reaction was analysed indirectly from the yellow product production over time at various initial catechol concentrations.

of Catechol

Results | Experiments indicate that the breakdown products to have a strong negative effect on cell survival.

product in whole cell cultures

Results | Comparing reaction profile graphs of cell cultures of XylE expressing cells Vs GFP-XylE expressing cells showed that the designed reporter enzyme has more than 10 fold decreased activity in comparison to wild type reporter enzyme catechol(2,3)dioxygenase.

in cell lysate

Results | Construnction of Michaelis-Menten curve and determination of kinetic parameters of catechol(2,3)dioxygenase enzyme in E.coli. The calculated Km value, the substrate concentration at which velocity is half of the maximum, is 0.71mM of catechol. The Vmax is 3.37mM/min at x20fold dilution of cell lysate.

in r.p.u.

Results | pVeg promoter in pSB1C3 vector, a high copy plasmid, has an 1.62 r.p.u value and in 3K3 vector, a low copy plasmid an 0.79 r.p.u. value. These values were derived by dividing signal from the production of HMS by the pVeg promoter population of cells by signal from the standard promoter J23101 (r.p.u value of 1).

We then hope to test each cell surface linker by adding TEV protease and measuring how much AIP is cleaved. The linker that gives us the highest degree of cleavage will be used for the final surface protein for cleavage by the Schistosoma elastase. The characterisation of the linkers in the [[Team:Imperial_College_London/Modules/Detection_Module | surface protein] are definitely an area for future work, as the surface protein has a vast number of potential applications. For more information, check out our new Software Tool!

However, this step would have been relatively straightforward and we have no reason to believe it would not function properly in B. subtilis, as like the organism from which the signaling system is taken (S. pneumoniae), it is Gram positive and has been shown to signal via AIPs in quorum sensing systems.