Part:BBa_K1590004

Regulator or Chromate responsive promoter

The protein encoded by this sequence is a putative chromate-responsive repressor of P</i>chr</i> (BBa_K1590003).

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

•••• iGEM Dundee 2015

This gene was found to produce a low level of stable product when expressed in E. coli cells. However, using an in vivo transcription/translation assay based on the promoter sequence of biobrick BBa_K1590003 it was not yet possible to show repressor or inducer activity of ChrB.

This sequence is found downstream of Pchr in the chrBACF operon in Ochrobactrum tritici 5bvl1, located in the transposable element TnOtChr. ChrB is suspected to inhibit the otherwise constitutive promoter Pchr in the absence of Cr(VI) by binding to an imperfect inverted repeat sequence located upstream of the initial ATG codon. Cr(VI) was expected to lift this repression, leading to the expression of the genes downstream of P</i>chr</i>.

The native ChrBACF - operon in Ochrobactrum tritici contained additional 3 additional downstream of P</i>chr</i>. chrA was found to express a chromate efflux pump, chrC and chrF were found to confer tolerance to superoxides. Deleting chrC and chrF was shown to have no significant effect on chromate resistance which at first seemed unusual, but considering that Cr(VI) is often reduced via Cr(V) and Cr(IV) intermediates, which can lead to the generation of superoxides, these 2 genes were suspected to act as a second line of defense.

Our experiments could not confirm a chromate dependent interaction of ChrB and Pchr. Our analysis focused on detection of ChrB and GFP as a fluorescent reporter, cloned downstream of Pchr individually, and in a strain, containing both plasmids.

When cloned individually, ChrB could be detected via a 6-His tag.

Figure 1: Western analysis of ChrB production from the Tat promoter BBa_K895000: Single colonies of JM110 pUniprom-chrB and JM110 pUniprom-chrB (opt) were used to inoculate 5 ml of LB broth supplemented with 100 ^µg/_ml ampicillin. 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 200 µl laemmli buffer, and the sample boiled for 10min. 10 µl of each sample was loaded on a SDS gel (12% acrylamide). pSB1C3 was included as a negative control, and hydA - 6-His as a positive control. Expected sizes: GFP ~ 27kDa, ChrB ~35kDa.

Figure 2: Comparison of presence of GFP in MG1655, containing all 4 combinations, pUniprom-chrB - pSB1C3-Pchr-gfp (A), pUniprom-chrB - pSB1C3-Pchr-gfp (B), pUniprom-chrB (opt) - pSB1C3-Pchr-gfp (A), and pUniprom-chrB (opt) - pSB1C3-Pchr-gfp (B). All samples were prepared and diluted to a suitable concentration for western blotting against GFP. pSB1C3, and pUniprom were included as a negative control, and PmanA-gfp as a positive control.

The same samples were used to blot against His-6 - ChrB as well, but no tagged protein could be detected.