Difference between revisions of "Part:BBa R0082:Experience"
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===Applications of BBa_R0082=== | ===Applications of BBa_R0082=== | ||
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+ | <h4><b><span><u>UCL 2015</u>: BBa_R0082 Testing</span></b></h4> | ||
+ | <p>The OmpR promoter ( <a href="https://parts.igem.org/Part:BBa_R0082">BBa_R0082</a>) from 2004 Antiquity was the part we wanted for our osmotic stress sensing device. To test if this promoter works, we ligated <a href="https://parts.igem.org/Part:BBa_R0082">BBa_R0082</a> with GFP reporter part, <a href="https://parts.igem.org/Part:BBa_I13504" target="_blank">BBa_I13504</a>, downstream of the promoter | ||
+ | , transformed it in DH5-α, and measured the fluorescence with a plate reader after being grown in LB containing varying concentrations of NaCl. | ||
+ | </p> | ||
+ | <br> | ||
+ | <div class="figure"> | ||
+ | <a href="https://static.igem.org/mediawiki/2015/5/54/OmpR_characterisation_corrected_to_0_NaCl.png" target="_blank"><img class="figurepic" src="https://static.igem.org/mediawiki/2015/5/54/OmpR_characterisation_corrected_to_0_NaCl.png"/></a> | ||
+ | <!--nitrosative stress, depression http://www.sciencedirect.com.libproxy.ucl.ac.uk/science/article/pii/S0149763411002120--> | ||
+ | <p><strong>Figure 3.</strong> Fluorescence of OmpR promoter-GFP DH5-α cultures when grown at varying concentrations of NaCl. | ||
+ | </p> | ||
+ | <br> | ||
+ | <br> | ||
+ | <p>Fig. 3 shows that at after around OD = 0.2, fluorescence becomes higher in the cultures containing NaCl compared to the culture with no epinephrine with the exceptions of 0.2% and 0.4% NaCl. However at OD=0.5, the culture with 0.2% NaCl started to fluoresce more than the 0% culture. | ||
+ | Overall, the cultures containing 0.05% and 0.1% had the highest amount of fluorescence, suggesting that at around 0.05% - 0.1% or perhaps a lower NaCl concentration, the OmpR promoter is induced at its optimal strength. | ||
+ | </p> | ||
+ | </html> | ||
===User Reviews=== | ===User Reviews=== | ||
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<figure> | <figure> | ||
<img width="50%" src="https://static.igem.org/mediawiki/parts/8/81/ORC_Fig.1.png"> | <img width="50%" src="https://static.igem.org/mediawiki/parts/8/81/ORC_Fig.1.png"> | ||
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<figcaption>Fig.1 <em>E.coli</em> TOP10 transformed PompC-RBS-RFP circuit</figcaption> | <figcaption>Fig.1 <em>E.coli</em> TOP10 transformed PompC-RBS-RFP circuit</figcaption> | ||
<img width="100%" src="https://static.igem.org/mediawiki/parts/6/65/ORC_Fig.2.png"> | <img width="100%" src="https://static.igem.org/mediawiki/parts/6/65/ORC_Fig.2.png"> | ||
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<figcaption>Fig.2 The result of sequencing</figcaption> | <figcaption>Fig.2 The result of sequencing</figcaption> | ||
</figure> | </figure> | ||
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<img width="50%" src="https://static.igem.org/mediawiki/parts/f/ff/ORC_Fig.3.png"> | <img width="50%" src="https://static.igem.org/mediawiki/parts/f/ff/ORC_Fig.3.png"> | ||
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<figcaption>Fig.3 Detecting the sensibility to light (from left to right: PompC-rfp, pSB1C3, rfp and RBS-rfp-ter)</figcaption> | <figcaption>Fig.3 Detecting the sensibility to light (from left to right: PompC-rfp, pSB1C3, rfp and RBS-rfp-ter)</figcaption> | ||
</figure> | </figure> | ||
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<img width="50%" src="https://static.igem.org/mediawiki/parts/b/bd/ORC_Fig.4.jpg"> | <img width="50%" src="https://static.igem.org/mediawiki/parts/b/bd/ORC_Fig.4.jpg"> | ||
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<figcaption>Fig.4 The results of the light sensitivity experiment (Fig.4A, from left to right: pSB1C3, RBS-rfp-ter, | <figcaption>Fig.4 The results of the light sensitivity experiment (Fig.4A, from left to right: pSB1C3, RBS-rfp-ter, | ||
PompC-rfp and rfp; Fig. 4B, from left to right: PompC-rfp with light,PompC-rfp without light; Fig. 4C, from left to right: PompC-rfp without light, rfp with light.)</figcaption> | PompC-rfp and rfp; Fig. 4B, from left to right: PompC-rfp with light,PompC-rfp without light; Fig. 4C, from left to right: PompC-rfp without light, rfp with light.)</figcaption> | ||
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<img width="70%" src="https://static.igem.org/mediawiki/parts/3/34/Bnu-ORC_Fig.5.png"> | <img width="70%" src="https://static.igem.org/mediawiki/parts/3/34/Bnu-ORC_Fig.5.png"> | ||
+ | <br/> | ||
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<figcaption>Fig. 5 0%,0.25%,0.50%,1% NaCl supplemented to the LBON medium</figcaption> | <figcaption>Fig. 5 0%,0.25%,0.50%,1% NaCl supplemented to the LBON medium</figcaption> | ||
</figure> | </figure> | ||
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With the osmolarity increasing, the expression of the rfp didn’t increase in experimental groups as we expected. That is to say under natural conditions, the expression of EnvZ-OmpR is too low to regulate the activity of PompC promoter. However, from the pictures we can see the colony of experimental groups still became red. It shows that the existing of EnvZ-OmpR makes the PompC promoter become a little bit active under the natural conditions, the basal activity of the PompC is correspondingly higher. So if we want to try to control the expression of the downstream target gene of the PompC by using EnvZ-OmpR-PompC circuit, we’d better knock out the EnvZ-OmpR gene in the engineering bacteria first. | With the osmolarity increasing, the expression of the rfp didn’t increase in experimental groups as we expected. That is to say under natural conditions, the expression of EnvZ-OmpR is too low to regulate the activity of PompC promoter. However, from the pictures we can see the colony of experimental groups still became red. It shows that the existing of EnvZ-OmpR makes the PompC promoter become a little bit active under the natural conditions, the basal activity of the PompC is correspondingly higher. So if we want to try to control the expression of the downstream target gene of the PompC by using EnvZ-OmpR-PompC circuit, we’d better knock out the EnvZ-OmpR gene in the engineering bacteria first. | ||
+ | |||
+ | |||
+ | |||
+ | ===Leaky Expression by the OmpR-Regulated Promoter on Different Vectors=== | ||
+ | (Characterized by SDU-Denmark) | ||
+ | |||
+ | <b>The leaky expression by the OmpR-regulated promoter is reduced when cloned into a low copy vector compared to a high copy vector.</b><br> | ||
+ | The expression properties of the OmpR-regulated promoter were investigated using a reporter system containing RFP under control of the OmpR-regulated promoter, <partinfo>BBa_M30011</partinfo>, was cloned into <i>E. coli</i> 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. <br> | ||
+ | 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 [https://www.ncbi.nlm.nih.gov/pubmed/16306980 Levskaya A] | ||
+ | |||
+ | <center> | ||
+ | https://static.igem.org/mediawiki/2017/7/79/T--SDU-Denmark--de-lyser-rodt.png | ||
+ | </center> | ||
+ | Figure 1. Fluorescence microscopy of RFP controlled by the OmpR-regulated promoter on a high copy vector in <i>E. coli</i> 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, [https://parts.igem.org/Part:BBa_J23114 BBa_J23114], and strong <partinfo>BBa_J23102</partinfo>constitutive 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 <i>E. coli</i> MG1655 and <i>E. coli</i> MG1655 ΔOmpR were studied to determine the baseline of the leaky expression not influenced by intrinsic pathways including the OmpR transcription factor. | ||
+ | <br> | ||
+ | |||
+ | |||
+ | <center> | ||
+ | https://static.igem.org/mediawiki/2017/c/c5/T--SDU-Denmark--Flow-celle1-reg.png | ||
+ | </center> | ||
+ | |||
+ | <b>Figure 2</b>. 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 <i>E. coli</i> 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.<br> | ||
+ | |||
+ | 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. <br> | ||
+ | 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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Latest revision as of 02:48, 2 November 2017
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_R0082
UCL 2015: BBa_R0082 Testing
The OmpR promoter ( BBa_R0082) from 2004 Antiquity was the part we wanted for our osmotic stress sensing device. To test if this promoter works, we ligated BBa_R0082 with GFP reporter part, BBa_I13504, downstream of the promoter , transformed it in DH5-α, and measured the fluorescence with a plate reader after being grown in LB containing varying concentrations of NaCl.
Figure 3. Fluorescence of OmpR promoter-GFP DH5-α cultures when grown at varying concentrations of NaCl.
Fig. 3 shows that at after around OD = 0.2, fluorescence becomes higher in the cultures containing NaCl compared to the culture with no epinephrine with the exceptions of 0.2% and 0.4% NaCl. However at OD=0.5, the culture with 0.2% NaCl started to fluoresce more than the 0% culture. Overall, the cultures containing 0.05% and 0.1% had the highest amount of fluorescence, suggesting that at around 0.05% - 0.1% or perhaps a lower NaCl concentration, the OmpR promoter is induced at its optimal strength.
User Reviews
UNIQ823aa71ac5335765-partinfo-00000001-QINU
No review score entered. Edinburgh iGEM 2009 |
|
UNIQ823aa71ac5335765-partinfo-00000003-QINU
UNIQ823aa71ac5335765-partinfo-00000004-QINU
- Characterization of new series of OmpC propmoters
Tokyo Tech iGEM2010 |
In order to characterize PompC(C) BBa_395301, PompC(CB) BBa395302 and PompC(CS1) BBa_395303, each promoter was attached to GFP and its transcriptional activity was measured through the GFP expression. [http://2010.igem.org/Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC ...see more about PompC series] |
UNIQ823aa71ac5335765-partinfo-00000005-QINU
Characterization of OmpC promoter by chemotaxis
UNIQ823aa71ac5335765-partinfo-00000006-QINU
XMU-China iGEM 2014 |
Figure 1. The plot of Moving radius versus Sucrose concentration. The four curves were measured after 10h, 11h, 12h and 16.5h respectively. Figure 2. Drawing horizontal line with 10% sucrose and erect line with water. Spotting cells on the cross. Two plates are parallel experiment. Culturing for 48 hours, we observed that reprogrammed E.coli has significant orientation to high concentration line. As high concentration sucrose generates high hyperosmosis, it has proved that CL-1 has the tendency swimming to high osmotic pressure.
We use semi-solid medium culture with gradient concentration of sucrose to characterize the device (BBa_K1412010). Setting the motile ability is proportional to the moving radius. From the plot, when no sucrose added in, the motile ability is the weakest. The motile ability keeps growing while the concentration of sucrose increased from 0 to 4%. Then the motile ability went down slightly as the sucrose concentration increased from 4% to 10%, but is still stronger than that at concentration 0. We can make the conclusion that our device is working as expectation, the motile ability went down because of the inhibition from hyperosmotic pressure. Besides, for even at the inhibiting osmotic pressure, the motile ability is still stronger than that without any inducer, reprogrammed CL-1 may even swim to killing osmotic pressure which will kill bacteria itself. |
UNIQ823aa71ac5335765-partinfo-00000007-QINU
More information, click here: [http://2014.igem.org/Team:XMU-China/Project_Application_BlackHole# XMU-China Black Hole]
Characterization of OmpC promoter of its basal activity UNIQ823aa71ac5335765-partinfo-00000008-QINU
BNU-CHINA iGEM 2015 |
We inserted PompC(BBa_R0082) into the upstream of the reporter gene rfp through tranditional cleavage and ligation method, which together is connected to vector pSB1C3 afterwards, and therefore we are able detect the basal activity of the promoter PompC, and the sensitivity to light and osmolarity.
1. The basal activity of PompC
We constructed the PompC-RBS-RFP circuit first (BBa_K1660005), when we just transformed this circuit into the E.coli Top 10, we wondrously found some of the colonies become red. It indicated these colonies had expressed RFP. It means without the regulation of the OmpR, the promoter PompC can start the transcription of downstream target gene. And then we sequenced these colonies which expressed RFP. The result indicated the PompC-RBS-RFP circuit did lead the expression of RFP.
We detected the light sensibility of the red colony. RFP coding device(BBa_J04450),RBS-rfp-terminator(BBa_K516032) and pSB1C3 were set as controls. We plated 100µL the overnight culture on LB medium+Chloramphenicol and cultivated them at 37°C. And half of them were under shading treatment. After 12 hours we observed the colonies.
The essence is that the EnvZ protein senses the mediun osmolarity and then forces the OmpR protein to take one of two alternative structures, which positively regulate OmpC synthesis.[2]
So we designed an experiment to detect under the normal level of the Envz, the trend of E.coli PompC activities with the change of osmotic pressure.
Overnight cultures of Top10 strains transformed with PompR-rfp, rfp, pSB1C3 and RBS-rfp-Ter respectively grown at 37°C in LB medium containing appropriated antibiotics were diluted at least 1:100 in the medium and incubated at 37°C as fresh cultures. After their OD600 reached 0.2~0.4, the fresh culture was diluted 1 : 3 into 4 ml of LBON medium(1g Tryptone, 1g Yeast Extract in 100mL H2O). For osmolarity conditions, the cultures were diluted with NaCl supplemented medium to the final concentration of 0%, 0.25%, 0.50% and 1%(wt/vol). After 12 hours of induction, the results are as follows.
With the osmolarity increasing, the expression of the rfp didn’t increase in experimental groups as we expected. That is to say under natural conditions, the expression of EnvZ-OmpR is too low to regulate the activity of PompC promoter. However, from the pictures we can see the colony of experimental groups still became red. It shows that the existing of EnvZ-OmpR makes the PompC promoter become a little bit active under the natural conditions, the basal activity of the PompC is correspondingly higher. So if we want to try to control the expression of the downstream target gene of the PompC by using EnvZ-OmpR-PompC circuit, we’d better knock out the EnvZ-OmpR gene in the engineering bacteria first.
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. <center> Figure 1. Fluorescence microscopy of RFP controlled by the OmpR-regulated promoter on a high copy vector in E. coli strain SØ928 ΔOmpR.
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. |
UNIQ823aa71ac5335765-partinfo-0000000F-QINU