"
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.
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.
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 500 μM of arsenite, and (b) 0, 10, 100, and 500 μM 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 500 μM, 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.
Salicylate
Our fluorescence assays confirm that the salicylate reporter part without a BamHI cutsite 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.Our controls for this assay were: blank LB, S. oneidensis with mtrB knocked out, S. oneidensis conjugated with an Anderson series promoter (0.1, 0.4, and 1.0) and mRFP, and a salicylate reporter strain without mRFP appended after mtrB. Relative fluorescence is reported using the same background subtraction and OD normalization described for the arsenic response assays.
Preliminary data of relative fluorescence at 1, 10, 100, and 500 μM of salicylate. Three strains of S. oneidensis are plotted: with mtrB knocked out, with an Anderson series promoter with strength 0.1 constitutively producing mRFP, and with our salicylate reporter with mRFP appended. Relative fluorescence is reported after normalization to optical density. For the testing strains (salicylate reporter w/o BamHI cutsite), fluorescence is averaged over 3 replicates, in addition to being averaged for each replicate individually over a time course of 7.5 hours.
Preliminary data strongly suggests that the salicylate reporter part without a BamHI cutsite 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. We are continuing testing with salicylate to examine more closely where promoter activity begins to increase, and to increase number of replicates to ensure statistical significance.
