Difference between revisions of "Part:BBa R0082:Experience"

No review score entered. Edinburgh iGEM 2009

We tested this promoter for use in our landmine-detection system. Our plan was to use this promoter together with a computationally designed TNT receptor and the hybrid Trz signal transduction protein, which has the EnvZ kinase domain, hence phosphorylates OmpR, activating this promoter, in the presence of TNT. To check that this promoter was working correctly, we attached it to GFP and assayed its activity in E. coli TOP10 cells as described on the Registry Measurement Page. (We also made a lacZ reporter version, BBa_K216010, so that we could do Miller assays as a backup, but at the time of writing we have not obtained good quantitative results from this). To induce the promoter, we used procaine, which activates the EnvZ kinase. Our results indicated that the promoter is activated by increasing levels of procaine from 0 to 15 mM, but there is very high basal activity under the conditions used, even in the absence of procaine. We also made an envZ mutant host strain using the KEIO collection of E. coli mutants, but at the time of writing, we have not yet repeated the tests using this host.

Tokyo Tech iGEM2010

Figure 1. Induction of new OmpC series in high osmolarity medium at 4 hours 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.

PompC(C)-GFPBBa_395304 , PompC(CB)-GFP BBa_395305, PompC(CS1)-GFP BBa_395306and PompC(WT)-GFPBBa_395307 on pSB3K3 was introduced into E. coli strain MG1655. A strain containing placI^q-GFP, plasmid (BBa_J54202), a constitutive GFP expressive promoter and promoterless GFP reporter plasmid (BBa_J54103) were used as a positive control and negative control respectively.

Overnight cultures of reporter strains grown at 37 °C in Medium A containing appropriated antibiotics were diluted at least 1:100 in the medium and incubated at 37 °C as fresh cultures. After their OD₅₉₀ reached 0.2, the fresh culture was diluted 1 : 3 into 2 ml of pre-warmed medium A. For high osmolarity conditions, the cultures were diluted with sucrose supplemented medium to the final concentration of 15% (wt/vol). After 4 hours of induction, fluorescence intensity was measured with fluorometer.

After 4 hours of high osmolarity induction by sucrose, transcriptional activity of PompC(CB)-GFP and PompC(CS1)-GFP increased 2.5 folds and 2.3 folds respectively. However, significant amount of leaky expression was found in PompC(CS1)-GFP without induction. In contrast, under the same conditions, we found no significant difference of GFP expression in PompC(C)-GFP and PompC(WT)-GFP. In addition, there was slightly higher GFP expression 1.7-folds occurred in the wild type at 2 hours of induction.

[http://2010.igem.org/Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC ...see more about PompC series]

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.

E.coli make use of the EnvZ/OmpR system to mediate signal transduction in response to environmental osmolarity changes. EnvZ, a histidine kinase, undergoes trans-autophosphorylation, then the high energy phosphoryl group is subsequently transferred to OmpR, a response regulator. In the system, OmpR-controlled promoter (PompC) is involved in. The expression strength of PompC is depending upon the medium osmolarity. When medium osmolarity is increasing, the EnvZ will phosphorylate more OmpR into phosphorylated OmpR (OmpR-P), and more OmpR-P will result in stronger expression strength of PompC. In our circuitry design, CheZ is upstream regulated by PompC. As the osmotic pressure is increasing, the motile ability of the engineered E.coli keeps growing, resulting in its suicide.

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.

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 (see parts page), 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.

2. The sensitivity of light

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.

We found all the plates transformed PompC-rfp and rfp become red. But the PompC-rfp colonies showed faintly red, and the differences with light or not are not obvious. It showes that PompC-rfp biobrick itself was not sensitive to light. And it also indicated the Pompc promoter has basal activities in E.coli TOP10. Because in nature, this promoter PompC is upstream of the ompC porin gene. The regulation of OmpC is determined by the EnvZ-OmpR osmosensing machinery. EnvZ phosphorylates OmpR to OmpR-P. At high osmolarity, EnvZ is more active, creating more OmpR-P. OmpR-P then binds to the low-affinity OmpR operator sites upstream of ompC.^[1]

3. The sensitivity of osmolarity for PompC

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.