Latest revision as of 09:18, 2 October 2024

LacZ generator driven by Plac

This is a β-galactosidase protein generator with strong RBS, driven by Plac promoter. LacI binds to the operator of Plac to inhibit β-galactosidase expression. IPTG can bind with LacI to induce β-galactosidase expression. With the different concentration of IPTG, it can express β-galactosidase at different levels.
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.

Sequence and Features

BBa_K4167660 is a β-galactosidase protein generator with strong RBS, 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.
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.

To construct the standard part, LacZ with RBS and promoter were 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 14h. 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.

Improvement by HSASNU 2023

1. New documentation to K4167660

K4167660 is a LacZ (β-galactosidase) generator driven by Plac. So, we added some documentation related to β-galactosidase. β-galactosidase has three enzymatic activities (Fig. 1). First, it can cleave the disaccharide lactose to form glucose and galactose, which can then enter glycolysis. Second, the enzyme can catalyze the transgalactosylation of lactose to allolactose, and, third, the allolactose can be cleaved to the monosaccharides. It is allolactose that binds to lacZ repressor and creates the positive feedback loop that regulates the amount of β-galactosidase in the cell [1].

Fig.1 Schematic summarizing the roles of β-galactosidase in the cell. The enzyme can hydrolyze lactose to galactose plus glucose, it can transgalactosylate to form allolactose, and it can hydrolyze allolactose. The presence of lactose results in the synthesis of allolactose which binds to the lac repressor and reduces its affinity for the lac operon. This in turn allows the synthesis of β-galactosidase, the product of the lacZ gene.

β-galactosidase is a tetramer of four identical polypeptide chains, each of 1023 amino acids. Within each monomer, the 1023 amino acids form five well-defined structural domains. The third (central) domain (residues 334–627) is a so called triose phosphate isomerase (TIM) or α8β8 barrel with the active site forming a deep pit at the C-terminal end of this barrel. As noted below, critical elements of the active site are also contributed by amino acids from elsewhere in the same polypeptide chain as well as from other chains within the tetramer [2].

Fig.2 The backbone structure of β-galactosidase tetramer. Domain 1, blue; Domain 2, green; Domain 3, yellow; Domain 4, cyan; Domain 5, red. Lighter and darker shading is used to differentiate equivalent domains in different subunits. Metal ions are shown as spheres, Na+, green; Mg++, blue.

The crystal structure of β-galactosidase was determined in an orthorhombic crystal with a single tetramer in the asymmetric unit [2].

Fig.3 Demonstration that β-galactosidase in crystals is catalytically active. Crystal of β-galactosidase (orthorhombic; ca.0.2 mm) in the absence (left) and in the presence, after about 2 h, of the substrate X-gal (right).

2. New data from HSASNU 2023 to K4167660

Since the part K4167660 is a β-galactosidase generator which can catalyze the substrate X-gal to generate blue product. To detect the part whether could express the functional enzyme or not, we cultured the BL21 cells transformed with the part K4167660 on the agar plate in the prsence of IPTG (0.5 mM) and X-gal (40 µg/mL). Some blue clones were observed (Fig.4).

Fig.4 The blue clones were observed on the agar plate after the part K4167660 was transformed into BL21 cells in the presence of IPTG and X-gal. A: Negative control without IPTG; B: experiment group with IPTG (0.5mM) and X-gal (40 µg/mL).

Refferences

[1] Juers DH, Matthews BW, Huber RE. LacZ β-galactosidase: structure and function of an enzyme of historical and molecular biological importance. Protein Sci. 2012; 21(12):1792-807. doi: 10.1002/pro.2165. Epub 2012 Nov 13.
[2] Juers DH, Wigley RH, Zhang X, Huber RE, Tronrud DE, Matthews BW. High resolution refinement of β-galactosidase in a new crystal form reveals multiple metal binding sites and provides a structural basis for a-complementation. Protein Sci. 2000, 9:1685–1699.

Improvement by ICJFLS 2024

1.New documentation of K4167660 collected from literatures

LacZ protein, also known as β-galactosidase, is an enzyme encoded by the lacZ gene of Escherichia coli. β-Galactosidase (β-gal) has been widely used as a transgene reporter enzyme. This enzyme can hydrolyze lactose and its derivatives (such as X-gal) to produce galactose and glucose. Due to its catalytic activity and easy detection characteristics, lacZ protein is widely used in molecular biology and genetic engineering, especially as a reporter gene.

1.1 The main structural characteristics of LacZ protein

①Tetrameric structure: LacZ protein is a homologous tetramer with a molecular weight of approximately 116 kDa per subunit. The four subunits interact to form a stable tetramer structure, which is essential for their catalytic activity.
②Active site: Each subunit contains an active site that can bind to substrates and catalyze reactions.
③Substrate binding pocket: A substrate binding pocket is formed around the active site, which specifically recognizes and binds lactose or its analogues.

Fig.1 Structural simulation diagram of LacZ protein.

1.2 The main functions of LacZ protein

LacZ protein has multiple important functions in lactose metabolism and molecular biology experiments in Escherichia coli:
①Lactose metabolism: In E.coli, lacZ protein hydrolyzes lactose to produce galactose and glucose, providing the carbon source and energy required for cellular metabolism.
②Reporter gene: Due to its catalytic activity, lacZ is often used as a reporter gene. For example, in the blue white screening, as a substrate, X-gal is hydrolyzed by lacZ to produce a blue product, which is used to distinguish between recombinant and non recombinant clones.
③Gene expression analysis: By measuring lacZ activity, the regulatory mechanism and level of gene expression can be studied.

1.3 LacZ protein activity assay method

The following methods are commonly used to study the activity and function of lacZ protein:
①ONPG decomposition experiment:
Principle: Using ONPG (ortho nitrophenyl-β-D-galactoside) as a substrate, lacZ catalyzes its decomposition to produce the yellow product ortho nitrophenol.
Step: React the cell lysate with ONPG and measure the absorbance changes using a spectrophotometer.
Result: The change in absorbance is directly proportional to the activity of lacZ.
②X-gal color rendering experiment:
Principle: X-gal generates blue insoluble products under lacZ catalysis, which are easy to observe with the naked eye.
Step: Cultivate bacteria containing the lacZ gene on a medium containing X-gal, and determine lacZ activity by observing blue colonies.
Result: Blue colonies indicate active expression of the lacZ gene.
③Fluorescent substrate experiment:
Principle: Using fluorescently labeled substrates (such as MUG), lacZ catalyzes the generation of fluorescent products.
Step: Mix the substrate with the sample and measure the fluorescence intensity using a fluorescence spectrophotometer.
Result: Fluorescence intensity is directly proportional to lacZ activity.

Fig. 2. Being a reporter gene, β-Gal staining in the whole brain and in the kidney of LacZ transgenic mice.
(A) (Left panel) The whole brain showed very strong lacZ expression. In the cere bellum, strong lacZ signals are present in granular layer and white matter. In the brainstem, strong lacZ expression is also detected. (Right panel) Enlargement of the area within the rectangle. (B) In the kidney, strong expression of lacZ was observed in the tubules, all the cells of the medulla, and the papillae of the kidney (left panel, kidney cross-section, right panel, enlarged view of medulla). Weaker expression was detectable in the pelvis, glomeruli, and blood vessels.

Refferences

[1] https://www.bilibili.com/read/cv36137131/?jump_opus=1
[2] Zhang GJ, Chen TB, Connolly B, Lin SA, Hargreaves R, Vanko A, Bednar B, Macneil DJ, Sur C, Williams DL. In vivo optical imaging of LacZ expression using lacZ transgenic mice. Assay Drug Dev Technol. 2009 Aug;7(4):391-9. doi: 10.1089/adt.2009.0195.

2.New data of BBa_ K4167660 collected from our lab

In our experiment, to identify whether the LacZ gene of recombinant plasmid express or not，both the complementary fragment of RNA aptamer and the plasmid pET-28a-Esch-lacZ were transformed into BL21DLacZ strain (with LacZ deletion). After culture 12h with fresh LB medium containing Kanamycin and Ampicillin, the LacZ protein (β-galactosidase) was purified using 6x His tag column for identification. The SDS-PAGE electrophoresis result was shown in Fig. 3.

Fig.3 The SDS-PAGE result shows expression and purification of LacZ protein.
M: Marker, 1: All supernatant proteins of BL21DLacZ strain, 2: All supernatant proteins of BL21DLacZ strain transformed with pET-28a-Esch-LacZ and the complementary fragment of E. coli aptamer, 3: Purified LacZ protein from the supernatant proteins of sample 2.