Part:BBa_K1660005

RFP controlled by the PompR promoter (express RFP under the OmpC promoter)

Introduction

PompC is a OmpR-controlled promoter which can be positively regulated by phosphorylated OmpR. This promoter is taken from the upstream region of ompC gene. Phosphorylated OmpR binds to the three operator sites and activates transcription. We inserted PompC(BBa_R0082) into the upstream of the reporter gene gfp 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 .

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 690
  Illegal AgeI site found at 802
  
• 1000
  COMPATIBLE WITH RFC[1000]

Results

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.

Fig.1 E.coli TOP10 transformed PompC-RBS-RFP circuit

Fig.2 The result of sequencing

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.

Fig.3 Detecting the sensibility to light (from left to right: PompC-rfp, pSB1C3, rfp and RBS-rfp-ter)

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.)

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]

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.

Fig. 5 0%，0.25%，0.50%，1% NaCl supplemented to the LBON medium

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.