Part:BBa_K1590003

Chromate responsive promoter

This promoter is found upstream of the ChrBACF - operon in Ochrobactrum tritici 5bvl1, located in the transposable element TnOtChr of 7189bp length. Pchr is suspected to be inducible by chromate via the chromate-responsive repressor ChrB.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 135
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

••• iGEM Dundee 2015

Dundee iGEM 2015 found this promoter to be active when used in E. coli cells.

Ochrobactrum tritici 5bvl1 was discovered in 2004 in a chromate contaminated wastewater treatment facility. A specific characteristic of this strain was that it could both, reduce Cr(VI) and be resistant to it at the same time. Previously characterised strains were either resistant or could reduce chromate. Over the last 10 years the group that discovered it started working out the underlying mechanisms of this set of characteristics.

The chrBACF - operon, expressed from the Pchr - promoter led to the expression of the downstream genes chrA, chrB, chrC, and chrF. chrA was found to express and efflux pump, whereas the gene product of chrB was identified as being a chromate-inducible regulator of Pchr. Additionally, chrC and chrF were found to confer tolerance to superoxides, which can be byproducts of the reduction of Cr(VI).

Pchr itself is believed to be regulated by the chromate-sensitive repressor chrB. This has made it a candidate for a biological sensor of chromate contamination in e.g. rivers (Branco et al.), or in milk (iGEM BIT 2013). Our project focused on hijacking this circuit in order to build a detector for stainless steels.

In our reporter construct, Pchr was cloned upstream of gfp. The construct was fully sequence confirmed and confirmed in size.

Figure 1: Gel for size confirmation of pSB1C3-Pchr-gfp. pSB1C3-Pchr-gfp (3) was found to contain the expected sequence.

When western blotting against GFP in the absence of ChrB, high concentrations of GFP could be detected.

Figure 2: Western analysis of GFP production driven by the chr promoter: Single colonies of JM110 + pSB1C3-Pchr-gfp (A) and MC1061 + pSB1C3-Pchr-gfp (B) were used to inoculate 5 ml of LB broth supplemented with 100 µg/ml chloramphenicol. After 16h of incubation at 37°C with agitation at 200rpm, each sample was subcultured into 5 ml of fresh, equally supplemented LB and cells were grown for 2 hours more. 1 ml of the subculture was then retrieved and pelleted. The pellet was resuspended in 1 ml TBS. 100 µl of the sample was mixed with 100 µl laemmli buffer, and boiled for 10min. 3 µl of each sample was loaded on a SDS gel (12% acrylamide). pSB1C3 was included as a negative control, and PmanA-gfp as a positive control.