Difference between revisions of "Part:BBa M30011:Experience"

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Applications of BBa_M30011

Stockholm 2015 iGEM Team

We wanted to use BBa_M30011 as part of an OmpR dependent regulatory circuit, which produces GFP at low osmolarity and RFP at high osmolarity (BBa_K1766004). First we decided to characterize how BBa_M30011 responds to changes in osmolarity.

We transformed two E. coli strains with BBa_M30011 in pSB1C3. One expressed wild-type EnvZ (TOP10) and the other had a ΔEnvZ mutation (BW25113). We then cultured both strains at different osmolarities, using minimal media substituted with 0%, 5%, 10% or 15% sucrose.

Results

Results of osmolarity test 1

The fluorescence was measured using a plate reader (Excitation:580+/-10 nm, Emission:627+/-30 nm).

Absolute fluorescence values were adjusted for optical density and normalized using the 0% samples.

Statistical analysis was done by Student’s t-test and standard error bars are shown in the chart.

‘*’ indicates significant P value.

ns: P > 0.05 (not significant).

'*' P ≤ 0.05.

'**' P ≤ 0.01.

'***' P ≤ 0.001.

'****' P ≤ 0.0001.

In the TOP10 strain a two fold increase in fluorescence could be seen in the high osmolarity condition (15%) compared to the low osmolarity (0%) condition. In the EnvZ deficient strain, however, there was only a 0.3 fold increase in fluoresence. The results show that BBa_M30011 is OmpR dependent and produces RFP in an osmolarity dependent manner.

Technion-Israel 2014 iGEM team

We used the biobrock BBa_M30011 (ompR controlled mRFP) to test the Taz construct (BBa_K1343016) we created. In addition, we decided to test the biobrick BBa_M30011 in response to changes in osmolarity.

Two isogenic strains of E. coli K12, BW25113 (parent strain from the Keio collection) and JW3367-3 (with ΔEnvZ mutation) were transformed with pSB1C3 carrying the BBa_M30011 reporter. The bacteria were cultured in growth media containing varying concentrations of NaCl. After two hours of growth the relative RFP fluorescence of the cultures was determined (fluorescence/OD).

As a positive control we used E. coli Top10 transformed with biobrick BBa_J04450 (RFP under Plac).

OD was measured at 600nm. Fluorescence excitation wavelength: 560nm Fluorescence emission wavelength: 612nm

Figure 1: Relative fluorescence dependent on NaCl concentration (mM)

We expected that the mutant strain (ΔEnvZ) would show a constant level of relative fluorescence which is lower than that of the parent strain. This is because histidine kinase protein which detects osmolarity changes in the cells environment (EnvZ) is not present in the mutant. The EnvZ does not phosphorylate the ompR. The low level of fluorescence could be due to another mechanism (such as an acetyl phosphate dependent mechanism) which phosphorylates the ompR, leading to activation of the PompC promoter. In Figure 1 we see that the mutant showed the expected constant low level of relative fluorescence.

Since the parent strain (+EnvZ) is sensitive to osmolarity changes in the cell’s environment, we expected that an increase in NaCl concentration would cause an increase in relative fluorescence. This is because at high osmolarity, more ompR is phosphorylated, leading to increased activation of the PompC promoter. However, the parent strain also showed a constant (but high) level of expression (see Figure 1). We repeated the experiment three times with different ranges and dilutions of NaCl concentration but all showed a similar result.

Figure 2: (A) E. coli BW25113 with no plasmid. (B) E. coli BW25113 containing ompR controlled mRFP (BBa_M30011) on pSB1C3. (C) E. coli JW3367-3 (ΔEnvZ) containing ompR controlled mRFP (BBa_M30011) on pSB1C3. (D) E. coli JW3367-3 (ΔEnvZ) with no plasmid.

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