Revision as of 19:30, 13 October 2022

Beta-galactosidase protein generator

Beta-galactosidase protein generator with strong RBS.

This part takes PoPS as input to express lacZ gene (BBa_I732005), encoding for beta-galactosidase enzyme. This enzyme can be used to cleave lactose molecule to glucose and galactose (see Fig.1), but can also be used as a reporter protein for colorimetric assays (together with X-Gal or ONPG as a substrate).

X-gal is cleaved by β-galactosidase yielding galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter is then oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue product (see Fig.2 and Fig.3).

Fig.1: lactose cleavage to glucose and galactose.

Fig.2: X-Gal cleavage to galactose and an insoluble blue product.

Fig.3: example of blue colonies bearing lacZ.

Sequence and Features

Improvement by ICJFLS2022

Overview:

β-galactosidase is encoded by LacZ gene. It is widely found in animals, plants, microorganisms and cultured cells. It can catalyze the hydrolysis of β-galactoside bonds in β-galactoside compounds and release free galactose. It can also cleave lactose to glucose and galactose.
β-galactosidase is often used as a reporter protein/enzyme for colorimetric assays. For example, β-galactosidase can cleave X-gal to yield galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter is then oxidized to 5-bromo-4-chloro Indigo, a blue color product which is easily measured.
In addition, β-galactosidase can decompose p-Nitrophenyl-β-D-Galactopyranoside to produce p-Nitrophenol. The product has a characteristic absorption peak at 400 nm. The activity of β-galactosidase can be characterized by the change of absorbance value, which is used to determine the activity of β-galactosidase.

BBa_K173004 is a β-galactosidase protein generator with strong RBS. To control the expression of β-galactosidase, we constructed BBa_K4167660 which is also a β-galactosidase protein generator with strong RBS, but it driven by Plac promoter. This promoter is mainly composed of Lac operon containing LacO site. LacI repressor, encoded by LacI gene, can bind with LacO site to inhibit the binding of RNA pol to the promoter, so the genes downstream expression is blocked. Serving as inducer, IPTG can bind with LacI inhibitor, making it detached from LacO site, which enables the transcription of downstream genes. So, the expression of β-galactosidase is regulated by IPTG induction. With the different concentration of IPTG, it can express β-galactosidase at different level. With the detection of p-Nitrophenol, the activity of β-galactosidase can be measured.

Results:

To construct the standard part, LacZ with RBS and promoter was checked for the restriction enzyme information, which is shown as follows:

Fig.1 The map of β-galactosidase generator described with SnapGene Viewer, showing the restriction enzyme information (no EcoRI and PstI sites).


After detecting the restriction enzyme information of β-galactosidase generator, it was inserted into the pSB1C3 plasmid to construct the standard part BBa_K4167660 with PCR method. Then it was identified as follows:

Fig.2 Identification of standard part BBa_K4167660, using PCR and digestion with EcoRI and PstI. M: Marker; 1: PCR result; Digestion result.


We compared the inducing effect of IPTG on the two β-galactosidase generators, using different concentration of IPTG. We set 5 groups: 2 groups of old generator(BBa_K173004) with or without IPTG and 2 groups of new generator(BBa_K4167660) with or without IPTG, and 1 negative control without β-galactosidase expression. At 0h, all groups’OD600 approximately reached to 0.6, then a certain of IPTG and p-Nitrophenyl-β-D-Galactopyranoside were added to the culture medium, incubated cells at 37℃ for 20h. Measure the absorption of OD400 and OD600 value for each group every 2h, using an automatic microplate reader. The results are showed as follows.

Fig.3 Inducing effect of IPTG on the two β-galactosidase generators. The OD400 value was standardized with OD600 value of each group at the same testing time. The figure indicated that BBa_K173004 expressing β-galactosidase was not affected by IPTG, while BBa_K4167660 expressing β-galactosidase was affected by IPTG. And different concentration of IPTG had the same inducing trend.


Then we detected the β-galactosidase expression with IPTG presence or absence, using both generators. The results showed that BBa_K173004 can express β-galactosidase whether IPTG was present or not, which means that BBa_K173004 expressing β-galactosidase was not affected by IPTG. However, BBa_K4167660 expressed β-galactosidase at a high level with IPTG presence, and it had some leakage expression without IPTG induction, which required further modification in future.

Fig.4 The comparison of β-galactosidase expression using BBa_K173004 with and without IPTG induction. The OD400 value is standardized with OD600 value of each group at the same testing time. The figure indicated that BBa_K173004 expressing β-galactosidase was not affected by IPTG.

Fig.5 The comparison of β-galactosidase expression using BBa_K4167660 with and without IPTG induction. The OD400 value is standardized with OD600 value of each group at the same testing time. The figure indicated that BBa_K4167660 expressed β-galactosidase at a high level with IPTG presence, and it had some leakage expression without IPTG induction.

References

1.Szabolcs Semsey, Sandeep Krishna.The effect of LacI autoregulation on the performance of the lactose utilization system in Escherichia col, Nucleic Acids Res 2013 Jul; 41(13): 6381–6390. 2.Adam J. Meyer, Thomas H. Segall-Shapiro, Emerson Glassey, Jing Zhang & Christopher A. Voigt. Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology, 2019, 15: 196–204.