Part:BBa_K4169021

pFrmR+FrmRAB - An operon that degrades formaldehyde

Basic Description

PfrmR is found upstream of the frmRAB formaldehyde detoxification operon. FrmR, the first product of the operon, is a member of the DUF156 family of DNA-binding transcriptional regulators. It binds the frmRAB promoter region and is negatively allosterically modulated by formaldehyde. FrmR is specific to formaldehyde. The genes frmA and frmB encode a formaldehyde dehydrogenase and S-formylglutathione hydrolase, respectively, and are responsible for detoxifying formaldehyde to formic acid in a glutathione-dependent pathway. The negative-feedback regulation of the frmRAB operon is similar to that of many other prokaryotic operons, whereby the transcription factor represses its own transcription.

Figure 1.Without formaldehyde

Figure 2.Containing formaldehyde

Origin

E.coli K12 MG1655.

Biology characteristic

1.This operon is induced by formaldehyde and degrades formaldehyde.

2.The protein expressed by FrmR can inhibit the upstream promoter, but formaldehyde can bind to frmR to prevent the inhibitory effect.

Experimental verification

Experiment 1

Culture：LB liquid medium +40%HCHO saturated aqueous solution.

Instrument: microplate reader.

The experimental group：DH5α with pFrmR+FrmRAB.

The control group：DH5α(No plasmid transformation).

Experimental steps

1.Pick the bacteria from the transformed plate and culture them in a bacterial bottle for 12-16h to test the colony OD600.

2.Transfer 100 μ L of bacterial liquid to 10mlLB liquid medium for about 7h.

3.Add different gradients of saturated formaldehyde solution (0,0.25, 1,2.5, 7.5, 25 μL).

4.200 μL of the mixture was added to the 96-well plate.

5.The microplate was set, the temperature was 37°, the absorbance was 600, and the overnight culture was detected every half hour.

Figure 3.The OD600 of colony in experimental group and control group changed with time

Figure 4.OD600 changes with time in experimental group and control group with 0.001% HCHO

When 0.25μL formaldehyde solution (0.001%) is added, the growth trend of the colony with FrmRAB operon is obviously in contrast to that of the bacteria without operon - the bacteria with operon grow better, while the bacteria without operon grow zigzag and drop rapidly after reaching the peak.

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 242
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 679
  Illegal AgeI site found at 1003
  
• 1000
  COMPATIBLE WITH RFC[1000]