Team:Imperial College London/Results/Exp4

Assaying the growth behaviour of E.coli Top10 cells transformed with either the XylE gene promoted by pVeg or alternatively as control, transformed with a CMR vector of identical size, we were able to determine if and what effect either catechol itself, or the breakdown product 2-hydroxymuconic semialdehyde had the expected growth-inhibiting effects on the cells.

Top10 cells were transformed with XylE and CMR respectively contained on the PSB1C3 vector. Experiments were performed both in LB and M9 medium. The 100ml wells were prepared to display an array of 0 to 2mM [Catechol] exposure. To determine cell-growth behavior we measured absorbance at 600nm using a POLARstar Omega plate-reader set to take measurements every 3 minutes over one hour, without catechol. Catechol was added to all wells after the pre-growth assay was completed and measurements of optical density were taken in 3 minute intervals over 3 hours.

Growth without Catechol

Pre-growth assays showed growth behavior as expected for cells growing normally.

Growth with Catechol

(LB) The addition of catechol had distinctive effects on the XylE expressing cells growing in LB medium. While at 0% catechol growth-behavior did not show a significant change (dark blue), even the lowest concentration of 0.25% catechol appeared to drastically reduce cell-survival (red). In contrast, CMR-control cells did not change their growing behavior in the presence of catechol.

From this we conclude that in LB medium, the breakdown product of catechol, 2-hydroxymuconic semialdehyde, has a lethal effect on E. coli.

(M9) Cells growing in M9 medium appeared more resistant to the effects of catechol. Even though absorbance at 380 nm increased significantly in well containing XylE expressing cells, indicating strong turnover of catechol by C2,3O (4), catechol did not appear to influence growing behavior in generalizable fashion: while a significant increase in the variance in the sample could be determined. Such was not observed for CMR expressing cells exposed to the same conditions. However, we were not able to establish a clear trend of growing-behavior in the presence of catechol as in case of the LB – XylE samples.

We conclude that the breakdown product of Catechol has a strong, deleterious effect on XylE expressing cells in an M9 medium, however, cells appear more able to resist the effects of 2-hydroxymuconic semialdehyde in M9 medium.

These results support the hypothesis that the breakdown product and not catechol itself has an intense deleterious effect on cell survival. This contributes to the safety of our system, as the output module is inherently coupled to the introduction to a substance lethal to the cells.