Part:BBa_M30011:Experience
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Applications of BBa_M30011
Stockholm 2017 iGEM Team
We designed an osmotic pressure test by the help of iGEM 2015 who already had worked with this biobrick (see below). Stockholm 2015 had shown that BBa_M30011 expressed more RFP when the cells were grown in higher concentrations of sucrose. However they only tested this at one OD value. We decided to test this at six different OD values. We had also found that UCL 2015 iGEM Team had worked with the same promoter that is in this biobrick (BBa_R0082). They had tested it with different NaCl concentrations but not got any conclusive results on how it worked. They concluded that they must have used too high NaCl concentrations. Thus we decided to test BBa_M30011 in different salt concentrations as well, and we used lower concentrations than UCL 2015 had used.
In the test we cultivated TOP10 cells in different sucrose and NaCl concentrations and measured the fluorescence of RFP at different OD values (0.1, 0.2, 0.3, 0.4, 0.5, 0.6). The results we got showed that OmpR produced more RFP as the osmotic pressure increased by sucrose, see figure 1-2. However for NaCl we saw no correlation between the increase in osmotic pressure and RFP expression, see figure 3-4.
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: 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: 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: 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.
- 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).
- 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.
Leaky Expression by the OmpR-Regulated Promoter on Different Vectors
(Characterized by SDU-Denmark)
The leaky expression by the OmpR-regulated promoter is reduced when cloned into a low copy vector compared to a high copy vector.
The expression properties of the OmpR-regulated promoter were investigated using a reporter system containing RFP under control of the OmpR-regulated promoter, BBa_M30011, was cloned into E. coli strain SØ928 ΔOmpR, lacking the OmpR transcription factor, on a high copy vector. By using a ΔompR strain, the background generated by stimulation of the intrinsic OmpR system is removed, and the strain functions as a negative control.
RFP expression was assessed by fluorescence microscopy using an Olympus IX83 with a photometrics prime camera, with exposure time for RFP at 200 ms.
Assessing the RFP expression by fluorescence microscopy, it was discovered that the OmpR-regulated promoter mediated gene expression even in the absence of its transcription factor, see Figure 1. This observation was confirmed by going through the literature [1].
Figure 1. Fluorescence microscopy of RFP controlled by the OmpR-regulated promoter on a high copy vector in E. coli strain SØ928 ΔOmpR.
On the basis of this finding, controlled gene expression by the OmpR-regulated promoter required a low copy plasmid or insertion into the chromosome. Protein expression of RFP in pSB1C3 with a copy number of 100-300 plasmids per cell, and pSB3K3 with a copy number of 10-12 plasmids per cell, was studied by flow cytometry. As for the determination of noise levels in the weak, BBa_J23114, and strong BBa_J23102constitutive promoters, the experiment was carried out in both LB medium and M9 minimal medium, the latter supplemented with 0.2% glycerol. In the LB medium, selection was carried out by the addition of 30 µg/mL chloramphenicol, 30 µg/mL kanamycin, or 50 µg/mL ampicillin, depending on the resistance, and for M9 minimal medium, the concentrations used were 60 µg/mL chloramphenicol, 60 µg/mL kanamycin, and 100 µg/mL ampicillin. Excitation of RFP was at 561 nm, and emission was measured around 580 nm. Expression levels in both E. coli MG1655 and E. coli MG1655 ΔompR were studied to determine the baseline of the leaky expression not influenced by intrinsic pathways including the OmpR transcription factor.
Figure 2. Flow cytometric fluorescence measurements in arbitrary units as a function of time. Left: Cultures were grown in LB medium. Right: Cultures were grown in M9 minimal medium supplemented with 0.2% glycerol. Fluorescence of RFP expressed by the the OmpR-regulated promoter on the high copy vector, pSB1C3, and the low copy vector, pSB3K3, in MG1655 WT and ΔOmpR MG1655 strain. All fluorescence levels were measured relative to the negative control WT E. coli MG1655, and the weak and strong constitutive promoters are included as references. Standard error of mean is shown, but are in several cases indistinguishable from the graph.
Fluorescence levels in the two different media display similar behavior, as seen in Figure 2. The main difference observed, was that the decrease in fluorescence over time was faster in LB medium than in M9 minimal medium, in concordance with the observations made in previous experiments. On a general level, the data revealed, that MG1655 cloned with the POmpR-RFP reporter system on the high copy vector exhibited a fluorescence level, equivalent to that mediated by the strong constitutive promoter. On the low copy vector, the POmpR-RFP reporter system yielded a fluorescence level comparable to the gene expression mediated by the weak constitutive promoter. On the other hand, expression levels in the MG1655 ΔompR strain were markedly reduced compared to MG1655, indicating that pathways including the transcription factor OmpR interfere with RFP expression under these conditions. Again, the fluorescence levels observed for the POmpR-RFP reporter system on the low copy vector were distinctly lower than for the high copy vector.
All things considered, the OmpR-regulated promoter was found to exhibit leaky expression comparable to the expression levels mediated by the constitutive promoters. When cloned into a low copy vector, the leaky expression was reduced prominently. Thus, to obtain proper regulation of gene expression by the OmpR-dependent promoter, a low copy vector is required.
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