Part:BBa_R0082:Experience
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Applications of BBa_R0082
User Reviews
UNIQea03489a0599e5c4-partinfo-00000000-QINU
No review score entered. Edinburgh iGEM 2009 |
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UNIQea03489a0599e5c4-partinfo-00000002-QINU
UNIQea03489a0599e5c4-partinfo-00000003-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] |
UNIQea03489a0599e5c4-partinfo-00000004-QINU
Characterization of OmpC promoter by chemotaxis
UNIQea03489a0599e5c4-partinfo-00000005-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. |
UNIQea03489a0599e5c4-partinfo-00000006-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 UNIQea03489a0599e5c4-partinfo-00000007-QINU
<I>BNU-CHINA iGEM 2015</I> |
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.
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. |