Team:Cornell/testing/project/wetlab/4/2

In order to characterize promoter activity in response to arsenic and salicylate, we appended mRFP downstream of mtrB in our reporter parts. We used these constructs to test the level of promoter activity at increasing arsenic and salicylate concentrations.

Control

In order to optimize experimental parameters, we began by measuring the fluorescence of mRFP with Anderson series promoters in Shewanella oneidensis MR-1. After adjusting the parameters of our tests to get a consistent response from control strains, we saw increasing relative fluorescence with increasing promoter strength. This suggests that the Anderson series constitutive promoters show similar activity in S. oneidensis as they do in E. coli. Relative fluorescence of four strains, normalized to optical density, averaged over 8 replicates after steady OD was reached and cells were in stationary phase. S. oneidensis with mtrB knocked out, and three strains of S. oneidensis expressing mRFP with Anderson series promoters strength 0.1, 0.7, and 1.0, were tested. mRFP fluorescence increases with increasing promoter strength.

Arsenic

We have begun characterization of our arsenic reporter’s response to arsenite and arsenate. In order to see if gene expression is increased in response to arsenite and arsenate, we measured fluorescence while varying concentration of arsenic-containing compounds in growth medium. We began by testing with arsenite because of the worry that an arsenate-reducing pathway native to S. oneidensis would add an unknown variable to tests using arsenate. All trials were run with the same controls: blank LB medium, S. oneidensis with mtrB knocked out, S. oneidensis conjugated with an Anderson promoter (0.1, 0.4, and 1.0) and mRFP, and an arsenic reporter strain without mRFP appended after mtrB. In the plots that follow, background fluorescence from LB was subtracted, and fluorescence normalized to optical density in order to obtain relative fluorescence per cell mass. Additionally, as described above in the control experiment, fluorescence data was averaged over a time course of 4.5 hours, after the cells had grown to a steady OD. Preliminary data of relative fluorescence at (a) 0, 10, 50, 100, and 500uM of arsenite, and (b) 0, 10, 100, and 500uM of arsenate. Four strains of S. oneidensis are plotted: with mtrB knocked out, with an arsenic reporter without mRFP appended after mtrB, and with two arsenic reporters with mRFP appended. Relative fluorescence is reported after normalization to optical density. For the two testing strains (arsenic reporter w/ BamHI and arsenic reporter w/o BamHI), fluorescence is averaged over 3 replicates, in addition to being averaged for each replicate individually over a time course of 4.5 hours.

Our preliminary data shows that as arsenite concentration increases from 0 to 500uM, relative fluorescence per cell mass increases by over two-fold for both reporter strains! Therefore, our arsenic reporters are responding to arsenite.

However, preliminary data from arsenate is somewhat more confounding – there is no clear trend in the current data. As S. oneidensis has native ability to reduce arsenates to arsenites, this could explain the lack of an obvious trend in fluorescence with increasing arsenate concentration.

As each graph is an average of only 3 replicates over 4.5 hour time courses, error bars are not included. We are currently continuing fluorescence assays to ensure statistical significance and to further define the dynamic range of our part.