Generator

Part:BBa_K173004

Designed by: Lorenzo Pasotti, Susanna Schiavi, Letizia Diamante, Paolo Magni   Group: iGEM09_UNIPV-Pavia   (2009-10-15)
Revision as of 19:18, 13 October 2022 by Emily212 (Talk | contribs)

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



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.

K4167660-fig.1.jpg


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:

K4167660-fig.1-2.jpg

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:

K4167666-fig.2.jpg

Fig.2 Identification of standard part pSB1C3-toehold switch-LacZ using PCR and digestion with EcoRI and PstI. M: Marker; 1: PCR result; Digestion result.


Toehold switch-LacZ plasmid was designed to express β-galactosidase controlled by the toehold switch and miRNA 34a-5p. It comprises the antisense sequence of miRNA 34a-5p, RBS, Linker and part sequence of miRNA 34a-5p, which form a toehold switch, as well as the gene of β-galactosidase. At the presence of miRNA 34a-5p, it binds to its antisense sequence, opening the toehold switch to trigger the expression of β-galactosidase which catalyzes the substrate X-gal to produce 5-bromo-4-chloro Indigo (blue color). The mechanism is shown as Fig.3.

K4167666-fig.3-2.jpg

Fig.3 The mechanism of toehold switch-LacZ.


To express β-galactosidase in BL21 bacteria, the recombined plasmid pET-28a-toehold switch-LacZ controlled by miRNA 34a-5p was constructed using PCR method. For identification, the restriction endonuclease digestion and PCR assays were performed, which showed that the fragment length of lacZ was consistent with the expected results (Fig.4)

K4167666-fig.4.jpg

Fig.4 Identification of pET-28a-toehold switch-lacZ plasmid. M: Marker, 1: The plasmid of pET-28a-toehold switch-LacZ, 2: The pET-28a-toehold switch-LacZ plasmid was digested by EcoRⅠ and Hind Ⅲ restriction endonuclease, 3: The LacZ gene amplified by PCR method.


pET-28a-toehold switch-lacZ plasmid was transfected into BL21ΔlacZ strain（LacZ deleted）. Under the optimal conditions, the cell-free expression system was prepared by mixing the cell extract with other components such as ATP, PEP, amino acid, etc. (see protocol section for details). After the filter paper was blocked with bovine serum albumin (BSA), washed and dried, a drop of the cell-free reaction system mentioned above fell onto the filter paper strip which was followed by putting it into the ultra-low temperature refrigerator and frozen dryer to form a paper strip sensor.

In order to obtain sensitive and fast detection effects, the reaction conditions that X-gal is converted to 5-bromo-4-chloro Indigo (blue color) catalyzed by β-galactosidase in the cell-free expression system was optimized under different temperature, reaction time and miRNA concentration, which were shown as follows:

K4167666-fig.5.jpg

Fig.5 The optimization of reaction temperature at which X-gal is converted to 5-bromo-4-chloro Indigo (blue color) catalyzed by β-galactosidase in the cell-free expression system. (A): OD570 value, (B): Photograph of paper strip sensor reaction in cell-free system.

K4167666-fig.6.jpg

Fig.6 The optimization of reaction time for β-galactosidase enzyme reaction in cell-free system. (A):OD570 value, (B): Photograph of paper strip sensor reaction in cell-free system.

K4167666-fig.7.jpg

Fig.7 The optimization of miRNA concentration to trigger the expression of β-galactosidase catalyzing the 5-bromo-4-chloro Indigo (blue color) production in cell-free system. (A):OD570 value, (B): Photograph of paper strip sensor reaction in cell-free system.

The optimization results showed that the best temperature is 30°C, as shown in Fig.5. When the reaction lasts for 1h, the reaction is almost over, so 1h is chosen as the best reaction time (Fig.6). For miR-34a-5p target sensor, the lowest limit of visible color development is 500fM (Fig.7).


References

1. Wan Y, Liu Y, Wang X, Wu J, Liu K, Zhou J, Liu L, Zhang C. Identification of differential microRNAs in cerebrospinal fluid and serum of patients with major depressive disorder. PLoS One, 2015 Mar 12;10(3): e0121975. doi: 10.1371/journal.pone.0121975
2. Zhou L, Zhu Y, Chen W, Tang Y. Emerging role of microRNAs in major depressive disorder and its implication on diagnosis and therapeutic response. J Affect Disord. 2021 May 1;286: 80-86. doi: 10.1016/j.jad.2021.02.063
3. Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: de-novo-designed regulators of gene expression. Cell. 2014 Nov 6;159(4):925-39. doi: 10.1016/j.cell.2014.10.002
4. Yakoh A, Pimpitak U, Rengpipat S, Hirankarn N, Chailapakul O, Chaiyo S. Paper-based electrochemical biosensor for diagnosing COVID-19: Detection of SARS-CoV-2 antibodies and antigen. Biosens Bioelectron. 2021;176:112912. doi:10.1016/j.bios.2020.11291


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