Part:BBa_K2936018

pFrmR-CAT

pFrmR is an engineered formaldehyde-inducible promoter, that expresses the genes when the concentration of CHOH in the environment reach a certain level. The catalase encoded by CAT gene could be reacted with TMB to make the culture blue.

Short description

Color reaction pathway.

Description

pFrmR is an engineered formaldehyde-inducible promoter, that expresses the genes when the concentration of CHOH in the environment reach a certain level. The catalase encoded by CAT gene could be reacted with TMB to make the culture blue.

Source

Escherichia coli

Design consideration

There is a leakage problem with the FrmR promoter, and the catalase is very sensitive with TMB.

Experiment

Introduction: pFrmR is an engineered formaldehyde-inducible promoter regulating the expression of target genes based on the concentration of H-CHO in the environment. CAT gene encodes catalase in the pathway, which could react with 3, 3’ ,5, 5’ -Tetramethylbenzidine (TMB) to generate blue color. Experiment:
A. The first stage:
(1) Culture E. coli harboring pFrmR - Cat gene in LB medium with different concentrations of formaldehyde (0 mg/ml, 10 mg/ml, 20 mg/ml, 40 mg/ml, 80 mg/ml, 100 mg/ml, 120 mg/ml) for 12 h .
(2) Measure the absorbance of the bacteria liquid at the wavelength of 600 nm and dilute the bacteria solution until the OD600 reached 0.5.
(3) Take 40 μl diluted bacteria liquid out in 96 - well plates .
(4) Add 160 μl TMB reagent in the bacterial liquid containing 96-well plate and incubate it at 37℃ for half an hour.
(5) Measure the absorbance of the reaction solution 96-well plate at the wavelength of 650 nm using ultraviolet spectrophotometer.

Data

The measured absorbance is shown below:

Figure 1.1 The original data of the A650 of the reaction solution

Form 1.1 The A650 of the reaction solution


Figure 1.2 Activation of pFrmR in different fromaldehyde concentrations

Analysis

When the E. coli were cultured with or without formaldehyde, they could react with TMB and the solution turned blue, which indicated that both of them produced catalase, and the absorbance at 650 nm was similar. The results indicated that FrmR promoter leakaged. There is no difference between the two situation because of the long culture time and the excessive amount of expressed enzymes, shortening the culture time can reduce the expression of enzymes, and whether the start-up of pFrmR can be seen.

The quantitative experiment

Considering to make the induction phenomenon more obvious by shortening the test culture time, we decided to take the bacterial liquid at specific times during the growth process and react with TMB. To avoid the effect on cell growth caused by sampling. We performed 32 culture batches and each group of measurements was repeated three times in parallel. We took E. coli containing pet-28b-cat plasmid as the control group, and E. coil with pet-28b-pfrmr-cat as the experimental group.

(1) Pick one colony of the strains into 5 ml LB medium and cultivate it overnight.
(2) Take 10 μl seed culture in 3 ml LB medim with 0 mg/ml, 80 mg/ml, and 120 mg/ml of formaldehyde.
(3) Take 200 μl cell culture at 3, 4.5, 5, 6, 7, 7.5, 8 and 8.5 hours respectively and stored at 4℃.
(4) Measure the absorbance of the bacteria liquid at the wavelength of 600 nm and dilute the bacteria solution until the OD600 reached 0.5.
(5) Take 40 μl diluted bacteria liquid out in 96 - well plates .
(6) Pick one colony of the strains into 5 ml LB medium and cultivate it overnight.
(7) Take 10 μl seed culture in 3 ml LB medim with 0 mg/ml, 80 mg/ml, and 120 mg/ml of formaldehyde.
(8) Take 200 μl cell culture at 3, 4.5, 5, 6, 7, 7.5, 8 and 8.5 hours respectively and stored at 4℃.
(9) Measure the absorbance of the bacteria liquid at the wavelength of 600 nm and dilute the bacteria solution until the OD600 reached 0.5.
(10) Take 40 μl diluted bacteria liquid out in 96 - well plates .
(11) Add 160 μl TMB reagent in the bacterial liquid containing 96-well plate and incubate it at 37℃ for half an hour.
(12) Measure the absorbance of the reaction solution 96-well plate at the wavelength of 650 nm using ultraviolet spectrophotometer.

Related conditions

Data

The measured absorbance is shown below:

Form 2 The absorbance of the reaction solution at the wavelength of 650 nm Analysis:
It can be seen from figure 1.3 that after cultivation for 4.5 h and 5 h in LB medium containing 80 mg/ml and 120 mg/ml formaldehyde, reaction of the bacterial liquid with TMB, the value of ABS650 are very similar to the value of positive control group.
This could indicate that although pFrmR exists promoter leakage phenomenon at the early stage of culture, the FrmR promoter was abled to induce by formaldehyde between 4h and 5h. Besides, the reason why the difference between the experimental groups was not obvious after 6h was that catalase might had been overexpressed and the detection agent TMB was extremely sensitive to Cat.

Usage and Biology

[1]Osman, D., Piergentili, C., Chen, J., Sayer, L. N., Uson, I., Huggins, T. G., Robinson, N. J., and Pohl, E. (2016) The Effectors and Sensory Sites of Formaldehyde-Responsive Regulator FrmR and Metal-Sensing Variant. [J]. Biol. Chem. 291, 19502-19516
Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1664
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1664
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 907
  Illegal XhoI site found at 1700
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1664
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1664
  
• 1000
  COMPATIBLE WITH RFC[1000]