Team:Imperial College London/Results

Results | Overlay of the spectra from the two cell cultures after catechol addition, reveals that in XylE expressing cultures a broad peak appears with maxima at 380nm

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 2-step amplification performing better than 1-step amplifier. The prediction is that for our system 2-step amplification output will be visible around 4 minutes earlier than the1-step amplification system

of C(2,3)O in whole cells

Results | Very useful information were acquired out of these assays of which the most important being the catechol(2,3)dioxygenase reaction profile with catechol. The graph generated delineate the course of the reaction in terms of yellow product production over time at various initial catechol concentrations

of Catechol

Results | Experiments indicate the breakdown product 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

Pveg promoter

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.