Team:SCUT-China A/Project Chromogenic-Reaction
Chromogenic Reaction
The β-galactosidase assay is used frequently in experiments of genetics, molecular biology, and other biological researches. [1] The amount of β-galactosidase released by the lysis E. coil can be detected using X-gal or oNPG, which forms a blue or yellow product after digested by β-galactosidase. We wanted to compare their effect and choose one of them to use in our project.
Ortho-Nitrophenyl-β-galactoside (oNPG) is a colorimetric and spectrophotometric
substrate for β-galactosidase. This compound is normally colorless. However, if β-galactosidase is present,
it will hydrolyze the oNPG into galactose and ortho-nitrophenol. The latter compound is yellow that can
be used to detect the enzymatic reaction by da colorimetric assay (at 420 nm wavelength). β-Galactosidase
is required for lactose utilization, so the intensity of the produced color can be used to measure of
the rate of enzymatic reaction .
Though oNPG mimics lactose and is hydrolyzed by β-galactosidase, it is unable
to act as an inducer for the lac operon. Without another lactose analog that can act as an inducer, such
as isopropyl β-D-1-thiogalactopyranoside (IPTG), β-galactosidase will not be transcribed and oNPG will
not be hydrolyzed.
X-gal is an analog of lactose. It can be hydrolyzed by the β-galactosidase, which cleaves the β-glycosidic
bond in D-lactose. X-gal, when catalyzed by β-galactosidase, will be cleaved into galactose and 5-bromo-4-chloro-3-hydroxyindole.
The latter product then spontaneously dimerizes and is oxidized into 5,5'-dibromo-4,4'-dichloro-indigo,
an insolubled intensive blue product. X-gal is colorless, so the presence of blue-colored product can
therefore be used to test the presence of β-galactosidase. This easy identification of the activity of
enzyme allows the β-galactosidase gene (the lacZ gene) to work as a reporter in various applications.[2]
Na2CO3
oNPG
x-gal
Z-Buffer
1. Grow the cells on LB medium+Chloramphenicol overnight.
2. translate cultures into LB medium+Chloramphenicol in another conical flask.
3. Incubate the cultures for about an hour.
4. Set your instrument to read OD600, measure OD600 of the cultures.
5. When OD600 reachs 0.4, add IPTG to the cultures.
6. Incubate the cultures for about half an hour.
7. Add the 5ml cultures into each cuvette from 200Ml cultures
8. Add Metal ion solution with these following concentration, each concentration has 2 replicates.
9. Incubate the cultures for about half an hour.
10. Set your instrument to read OD600, measure OD600 of the cultures.
11. Centrifuge at 15000g, 15mins.
12. In 96 hole plate，set up a reaction mix at room temperature containing:
oNPGreaction mix
Z-Buffer (includedβ-mercaptoethanol )
oNPG
Lysate supernatant
X-gal reaction mix
Z-Buffer (includedβ-mercaptoethanol )
X-gal
Lysate supernatant
13. Add Na2CO3, and measure your samples by a plate reader (OD600 and OD420, OD550).
β-galactosidase activity /unit =1000 ×[(Abs420-1.75 × Abs550)/(t ×V× Abs600)]
T/min is the reaction time after adding oNPG to the reaction substrate
V/ml is the volume after sample dilution
14. Take pictures of the plate.
15. Remarks: β-galactosidase activity /unit is defined as the amount of 1umol oNPG substrate hydrolyzed at 37℃ in 1 min.
In order to figure out which one have a more obvious effect, oNPG or X-gal, first we found out the best response range of our devices. Then we compared the effect between oNPG and X-gal.
As our expectation, the amount of β-galactosidase that hydrolyzes the oNPG into yellow and hydrolyzes x-gal into blue released by the lysis of E. coil is linked to the concentration of metal ions. The system induced by different concentrations of metal ions showed different enzyme activity, which is reflected in the shading of the color of the chromogenic reaction.
The result indicated that the shading of the blue product produced by X-gald is more obvious than yellow product produced by oNPG. Although, oNPG is widely used in measuring enzyme activity of β-galactosidase, it needs to measure its abs420 which is difficult to achieved by smart phone. Considering all of the factors, we chose X-gal to measure the amount of β-galactosidase, whose product have an obvious difference in color shading among different concentrations.
With the use of X-gal, we measured the LOD and range of detection. Then we compared it to the Hygienic standard for drinking water and wastewater discharge in China.
The following tables the limit of detection of this method. And our LOD is lower than wastewater discharge standard (China)
| Metal ions | LOD (mol/L) | Range of detection | Hygienic standard for drinking water (China) | wastewater discharge standard (China) |
|---|---|---|---|---|
| Cd | 10-6 | 10-6~~10-5 | 4.4 X 10-8 | 8.9 X 10-7 |
| Hg | 2 X 10-8 | 2 X 10-8~~10-7 | 5 X 10-9 | 2.5 X 10-7 |
| Pb | 5 X 10-7 | 5 X10-7~~10-5 | 5 X 10-8 | 5 X 10-6 |
Table 1. the data of detecting devices' effect
According to our experiments, we chose x-gal to measure the amount of β-galactosidase. There was no doubt that our detecting devices have a strong ability to detect the metal ions, through the release of β-galactosidase caused by the lysis of E. coil. Inspiritingly we discovered that the shading of the blue has an obvious contact to metal ions' concentration. By detecting the shading of the product' color, we can qualitatively detect the concentration of different metals.
On next stage, we would build up a model and write an application of smart phone for detecting the concentration of different metal ions based on the result.
[1]. Alexander J. Ninfa, Alexander J. (2009). Fundamental Laboratory Approaches for Biochemistry and Biotechnology. ISBN 978-0-470-47131-9.
[2]. Sandhu, Sardul Singh (2010). Recombinant DNA Technology. I K International Publishing House. p. 116. ISBN 978-9380578446.
