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

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Applications of BBa_M30011

Stockholm 2017 iGEM Team

Before we ligated the OmpR promoter to sialidase we tested how the OmpR promoter works. We found an already existing biobrick for this (BBa_M30011) that had OmpR connected to RFP as a reporter. We designed an osmotic pressure test by the help of iGEM 2015 who already had worked with this biobrick. In the test we cultivated TOP10 cells in different sucrose and NaCl concentrations and measured the fluorescence of RFP at different OD points (for more detailed explanation see the experience page for BBa_M30011). The results we got showed that OmpR produced more RFP as the osmotic pressure increased by sucrose, see figure 4-5. However for NaCl we saw no correlation between the increase in osmotic pressure and RFP expression, see figure 6-7. We therefore decided to only use sucrose when we tested our final biobrick.

Figure 2

Figure 2: Expression of RFP fluorescence after activation of the osmotic pressure sensitive promoter OmpR with different sucrose concentrations. As the sucrose concentration increases, and by so the osmotic pressure rises, the RFP expression is increased.

Figure 3

Figure 3: Expression of RFP fluorescence plotted against the OD-value at which the sample was taken. The higher sucrose concentrations did not reach the highest OD-values and are therefore missing some points.

Figure 4

Figure 4: Expression of RFP fluorescence after activation of the osmotic pressure sensitive promoter OmpR with different NaCl concentrations. No correlation can be seen from this graph.

Figure 5

Figure 5: Expression of RFP fluorescence plotted against the OD-value at which the sample was taken. No correlation can be seen from this graph.

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.

Osmolarity Test I: BBa_M30011 in wild-type and ΔEnvZ E. coli

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 cultured both strains at different osmolarities, using minimal media substituted with 0 %, 5 %, 10 % or 15 % sucrose.

Figure 3

• 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.

As seen in Figure 3, 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 fluorescence.

The results indicate that BBa_M30011 is controlled by OmpR and produces RFP in an osmolarity dependent manner.

Osmolarity Test II: BBa_M30011 in pSB3C5 low copy number plasmid

We wanted to characterize BBa_M30011 further, in order to have more reliable results. We therefore cloned the BioBrick into pSB3C5, a low copy number plasmid. The osmolarity test for TOP10 E. coli was repeated, this time using LB media supplemented with sucrose. The data obtained was then compared to the TOP10 data from Osmolarity Test I (see Figure 4).

Figure 4

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

• Absolute fluorescence values were adjusted for optical density.

• 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.

As expected, the pSB3C5 samples were less fluorescent than the pSB1C3 samples. The fold change in fluorescence, however, remained roughly the same for the high and low copy number plasmids. Interestingly, the standard deviations were much smaller in the pSB3C5 samples. The statistical analysis also showed that the data was more significant.

It is possible that using a low copy number plasmid disturbs the stoichiometric conditions of the cell less than using a high copy number plasmid. The cells are probably also less stressed when cultured in LB media, compared to minimal media. Both these factors probably contributed to the more even and reliable results.

We conclude that this part functions as described by the makers. Furthermore we recommend using it together with a low copy number plasmid to have more consistent results.

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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