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.

Figure 5.At the same time, under different concentrations of formaldehyde, the OD600 difference between the experimental group and the control group.The X-axis is incubation time,the Y-axis is formaldehyde at different concentrations.The darker the color, the greater the difference between the experimental group and the control group. On the other hand,the lighter the color, the smaller the difference.

Different concentrations of formaldehyde were added to the experimental group and the control group at the same time, and the bacteria were placed in the microplate reader for overnight culture and real-time monitoring of the OD value of the colony.There was no difference in the growth trend between the experimental group and the control group for other gradient formaldehyde solutions, and almost all of them showed a downward trend. It was proved that colonies containing the FrmRAB operon and colonies without the FrmRAb operon could not adapt to growth in formaldehyde solution higher than 0.25ul. But it's also possible that we didn't cultivate it long enough.

Experiment 2

Pick the bacteria on the transformed bacterial plate and culture them in the bacterial bottle for 12-16h

Culture：LB liquid medium +40%HCHO saturated aqueous solution + sodium sulfite fuchsin mixture

The experimental group：DH5α with pFrmR+FrmRAB

The control group：DH5α(No plasmid transformation)

Experimental steps

1.Six tubes of 100 microliters of bacterial liquid were taken from the experimental group and the control group, and 10mlLB liquid medium was added to the culture for about 7 hours

2.Different gradients of saturated formaldehyde were added（0，0.25，1，2.5，7.5，25μL）

3.Prepare sodium sulfite fuchsin solution (sodium sulfite 2.5g/l fuchsin 0.5g/l)

4.Add the same amount of sodium sulfite fuchsin solution to all samples and observe the color change

Figure 6.+ 0.25μl 40%HCHO(0.001%), no operon gene on the left, FrmRAB operon gene on the right, same as the following figure

Figure 7.+1μL40%HCHO(0.004%)

Figure 8.+2.5μL40%HCHO(0.01%)

Figure 8.+7.5μL40%HCHO(0.03%)

Figure 8.+25μL40%HCHO(0.1%)

In the +0.25μL40%HCHO group, the red color of the bacteria solution containing FrmRAB operon was not as deep as that of the control group, indicating that formaldehyde dehydrogenase effectively degraded part of formaldehyde. However, the sodium sulfite fuchsin solution was very sensitive to formaldehyde color development, so it did not appear completely colorless. The same is true for a group of +1 μl 40%HCHO. However, for the formaldehyde solution with higher concentration, the color of the experimental group and the control group was almost no difference, which proved that the pFrmRAB operon did not adapt to the formaldehyde solution with higher concentration.

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]