Part:BBa_I732005

lacZ (encoding beta-galactosidase, full-length)

LacZ encodes beta-galactosidase, an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose. Beta-galactosidase can also cleaves X-gal and ONPG into colorful products.

Added by THINKER_CHINA

β-galactosidase

The chromogenic module is the key to the final presentation of our detection system's result. And the chromogenic module's results provide us with fundamental indicators for the qualitative and quantitative analysis of the subject. In our final product, the chromogenic module and the lysis module work together, but when verifying the chromogenic module alone, we use the engineered strain containing only the chromogenic module for verification. Furthermore, we used the arabinose promoter to induce β-galactoside's gene structures to avoid other factors influence.

The principle of the chromogenic module in our product is the chromogenic reaction between x-gal and endogenous bacterial β-galactosidase, which would produce the color blue. And the higher the concentration of the test substance, the greater the number of bacteria-induced to lyse will be. Consequently, there will also be more β-galactosidase participation in the chromogenic reaction, which causes results of color blue accumulation resulting in a darker gray value. Therefore, whether the chromogenic module presents the color blue as the result or other colors, it can still express the qualitative detection of the factor to be detected and whether it contains the element to be detected. On the other hand, if the module successfully displays the color blue, the gray value of the blue color can clearly show the density of the subject being tested. Furthermore, since x-gal can penetrate the bacterial cell membrane, it can react directly with its endogenous ß-galactosidase without lysis of the bacteria. Therefore, to ensure that all chromogenic reaction results are carried out by the ß-galactosidase released after the bacteria receive the signal to lysis, we will centrifuge the solution and take the supernatant for verification after waiting for the gene expression reaction.

The experimental steps of our verification experiment of the color rendering module are as follows:

1. Cultivate the engineered strain in the LB medium and wait until its OD600 value (surface bacterial concentration) reaches 0.4.

2. Add different concentrations of arabinose to the culture as a signal factor to initiate gene expression for more than half an hour.

3. Lyse the bacteria by sonication and take the supernatant after centrifugation for subsequent procedures.

4. Add x-gal and allow reaction for half an hour.

5. Observe the change of OD620 (absorbance value at 620 nm of the light wave) with a microplate reader.

Figure 1

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]

Team: SMS_Shenzhen 2022

Description of the structure of E.coli beta-galactosidase

The β-galactosidase tetramer (Fig. 1) is comprised of four polypeptide chains, labeled A–D, each of 1023 amino acids.[1] Each 1023-amino-acid monomer is made up of five domains, 1–5, which are respectively colored blue, green, yellow, cyan and red in Fig. 1. Domain 3 has an α/β or ‘TIM’ barrel structure with the active site located at the C-terminal end of the barrel.[2] Both Mg2+ and Na+ are required for maximal activity of β-galactosidase.[3]

Fig.1 | View of the β-galactosidase tetramer looking down one of the two-fold axes. Coloring is by domain: complementation peptide, orange; Domain 1, blue; Domain 2, green; Domain 3, yellow; Domain 4, cyan; Domain 5, red. Lighter and darker shades of a given color are used to distinguish the same domain in different subunits. The metal cations in each of the four active sites are shown as spheres: Na+, green; Mg++, blue. [2]

References

[1] Matthews, Brian W. "The structure of E. coli β-galactosidase." Comptes rendus biologies 328.6 (2005): 549-556.
[2] D.H. Juers, R.H. Jacobson, D. Wigley, X.-J. Zhang, R.E. Hu- ber, D.E. Tronrud, B.W. Matthews, High resolution refine- ment of β-galactosidase in a new crystal form reveals mul- tiple metal-binding sites and provides a structural basis for α-complementation, Protein Sci. 9 (2000) 1685–1699.
[3] K. Wallenfels, R. Weil, in: β-Galactosidase, third ed., in: The Enzymes, vol. 7, Academic Press, London, 1972, pp. 617–663.