"
Team:Cornell/testing/project/wetlab/4/2
From 2012.igem.org
-
Wet Lab
- Overview
- Chassis
- DNA Assembly
- Testing & Results
- Future Work
- Animation
Fluorescence
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
In order to determine if gene expression is increased in the presence of arsenic, we inoculated our growth medium with varying concentrations of either arsenite or arsenate and measured fluorescence using the BioTek Instruments Synergy™ HT Multi-Mode Microplate Reader. Trials were run using blank LB medium and JG700 as negative controls, while conjugated JG700 strains with Anderson promoters (0.1, 0.4, 1.0) upstream of mRFP were used as positive controls. Background fluorescence from LB was subtracted and fluorescence normalized to optical density in order to obtain relative fluorescence per cell mass. 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 0, 10, 50, 100, and 500 μM of (a) arsenite, and (b) arsenate. Relative fluorescence is reported after normalization to optical density.
Our preliminary data shows that as arsenite concentration is increased, relative fluorescence increases by over two-fold in both reporter strains! Therefore, our arsenic reporters are responding to arsenite.
However, our preliminary data from arsenate treatment shows no clear trend. As S. oneidensis has the native ability to reduce arsenates to arsenites, this may contribute to the lack of an obvious trend.
Because relative fluorescence 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 constructs.
Salicylate
Our fluorescence assays confirm that the salicylate reporter construct without a BamHI cut-site, SAL2, responds to salicylate in a range of 10-100 μM. As with tests characterizing response to arsenites and arsenates, we measured fluorescence while varying concentration of salicylate in LB medium. Trials were run using blank LB medium and JG700 as negative controls, while conjugated JG700 strains with Anderson promoters (0.1, 0.4, 1.0) upstream of mRFP were used as positive controls. Background fluorescence from LB was subtracted and fluorescence normalized to optical density in order to obtain relative fluorescence per cell mass. Fluorescence data was averaged over a time course of 7.5 hours, after the cells had grown to a steady OD.
Preliminary data of relative fluorescence at 1, 10, 100, and 500 μM of salicylate. Relative fluorescence is reported after normalization to optical density.
Preliminary data strongly suggests that SAL2 responds to salicylate at concentrations in the order of hundreds of μM. Additionally, comparison to fluorescence from the Anderson 0.1 promoter (not shown in graph) suggests that the induced promoter activity may have similar strength to 0.1 on the Anderson series scale.
SAL2 fluorescence is averaged over 3 replicates in addition to averaging replicate individually over a time course of 7.5 hours. We are continuing to characterize our salicylate reporter strains.
