Difference between revisions of "Part:BBa K2936014"

Latest revision as of 03:50, 22 October 2019

cat(encoding catalase）

This is one of catalases from Parageobacillus.

Short description

An enzyme that catalyzes the reduction of hydrogen peroxide.

Description

Catalase is a enzyme that catalyzes the decomposition of hydrogen peroxide to water and oxygen.

Source

Parageobacillus.

Design consideration

CAT gene encodes catalase in the pathway, which could react with 3, 3’ ,5, 5’ -Tetramethylbenzidine (TMB) to generate blue color.

Mechanism

The qualitative experiment

(1) Pick one colony of E. coli harboring Cat genethe into 5 ml LB medium and cultivate it overnight. (2) Measure the Aorbance of the bacteria liquid at the wavelength of 600 nm and dilute the bacteria solution until the OD600 reached 0.5. (3) Make 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 Aorbance of the reaction solution 96-well plate at the wavelength of 650 nm using ultraviolet spectrophotometer.

Data

Analysis

From the figure 14.1, the color of the third group is enough obvious compared to the background. And at the same time, in order to balance the principle of economy, the ratio of bacterial culture (OD650=0.5) to TMB is designed to be 1:4 .

Results:

The numbers on the photo are the the concentration of catalase ( μl / ml ).

Related conditions

Data

The experimental data was derived from four parallel experiments.

Analysis

From the figure 14.2, the catalytic reaction of CAT with TMB is an obvious color reaction, and the reaction solution appears blue color under natural light. What’s more, it can be seen that as the proportion of catalase getting up, the color of the reaction solution is getting darker and the value of A650 is getting larger. From the figure 14.5, when the ratio of catalase at the range from 2.5E-03 to 3.0E-02 (μl/ml), the concentration of catalase ( μl / ml ) and the value of A650 can be represented by the relation A650=13.872Ccatalase+0.0224. This proves that the CAT gene can be used as an reporter gene for qualitative characterization, as the characteristics they confer on E.coil expressing catalase are easily identified and measured, and they are selectable markers.

Sequence and Features