Blue White Screening use in biotechnology

Biotechnology students

1. Blue White Screening Instructor: Aqdas Maria Razzaq

2. Principle The method relies on the use of a plasmid vector that contains the lacZ gene, which encodes the enzyme β-galactosidase. The lacZ gene is under the control of the lac operon, and the enzyme it encodes can cleave specific substrates, such as X-gal, to produce a visible color change. If foreign DNA is inserted into the MCS, it disrupts the lacZ gene, preventing the production of functional β-galactosidase . If no DNA is inserted, the lacZ gene remains intact and fully functional.

3. Components Required Plasmid Vector: The vector used in blue-white screening includes: The lacZ gene, which encodes β- galactosidase.A multiple cloning site (MCS) located within the lacZ gene. The MCS provides several restriction enzyme sites where foreign DNA can be inserted. X-gal : A colorless, synthetic substrate for β- galactosidase. When cleaved by this enzyme, X-gal produces a blue-colored product, 5,5'-dibromo-4,4'-dichloro-indigo . Inducer: Isopropyl β-D-1-thiogalactopyranoside (IPTG) is used along with X-gal for blue-white screening. IPTG is a non- metabolizable analog of galactose that induces the expression of lacZ gene.

4. Purpose of X-Gal Substrate X-gal (5-bromo-4-chloro-3-indolyl- β- D- galactopyranoside ) is used in blue-white screening, a method commonly employed in molecular biology to distinguish between recombinant and non-recombinant bacterial colonies. Substrate for β- galactosidase : X-gal is a synthetic substrate for the enzyme β- galactosidase, which is encoded by the lacZ gene. When β- galactosidase cleaves X-gal, it produces a blue-colored compound called 5,5'-dibromo-4,4'-dichloro-indigo

5. Characteristics of plasmid Vectors Plasmids are commonly used as vectors in molecular biology and genetic engineering to carry and deliver foreign DNA into host cells. An effective plasmid vector must have specific characteristics that enable it to function efficiently; Origin of Replication (Ori ) Selectable Marker Genes (antibiotic resistant gene) Multiple Cloning Site (Restriction Digestion sites) Small Size (5-25kb) High Copy Number Promoter Regions (Important for expression vector) Compatibility with Host Cells

6. Method of Blue White Screening Identification of Recombinant Bacteria Blue-white screening is a rapid and efficient technique for the identification of recombinant bacteria. It relies on the activity of β-galactosidase, an enzyme occurring in  E. coli , which cleaves lactose into glucose and galactose. Disrupting the LacZ Gene The presence of lactose in the surrounding environment triggers the lacZ operon in  E. coli . The operon activity results in the production of β- galactoisdase enzyme that metabolizes the lactose. When the plasmid vector is taken up by such cells, a functional β- galatosidase enzyme is produced. A multiple cloning site (MCS) is present within the lacZ sequence in the plasmid vector. This sequence can be nicked by restriction enzymes to insert the foreign DNA. When a plasmid vector containing foreign DNA is taken up by the host  E. coli , the α-complementation does not occur, therefore, a functional β-galactosidase enzyme is not produced.

7. How Does Blue White Screening Work? For screening the clones containing recombinant DNA, a chromogenic substrate known as X-gal is added to the agar plate. If β-galactosidase is produced, X-gal is hydrolyzed to form 5-bromo-4-chloro-indoxyl, which spontaneously dimerizes to produce an insoluble blue pigment called 5,5’-dibromo-4,4’-dichloro-indigo. The colonies formed by non-recombinant cells, therefore appear blue in color while the recombinant ones appear white. The desired recombinant colonies can be easily picked and cultured. It should be noted that IPTG is not a substrate for β-galactosidase but only an inducer. For visual screening purposes, chromogenic substrate like X-gal is required.

8. Bacterial Plasmid Vector Cloning

9. Protocol Of Blue White Screening X-Gal and IPTG incorporated into agar media before pouring into plates or added onto pre-made plates. Prepare 20 mg/ml X-Gal in DMF ( Dimethylformamide ) act as an excellent solvent Prepare 100mM IPTG solution in dH2O or dilute from a 1M IPTG solution. This solution is stable for 1 year . Screening on agar media containing IPTG and X-Gal. Autoclave the growth media agar, then cool to 50°C . Add 10 µl of 20 mg/ml X-Gal solution per 1 ml of media or 2 µl of 100 mg/ml X-Gal solution per 1 ml of media. Add 10 µl IPTG (100mM) per 1 ml of media for a final concentration of 1mM. Add the screening antibiotic. Pour plates and allow them to cool to room temperature before use. This usually takes at least 30 minutes. Spread transformed competent cells as desired. Screening on pre-made agar plates lacking IPTG and X-Gal as a control. Pour autoclaved growth media containing screening antibiotic on media plates and dry in a laminar flow hood. Add 40 µl 100mM IPTG and 120 µl X-Gal (20 mg/ml) to the surface of each plate and spread over the entire surface. Dry X-Gal/IPTG-coated media in a laminar flow hood for approximately 30 minutes before use. Spread transformed competent cells and incubate inverted at either 37°C until blue colonies form (usually ~24 hours).

10. Result Interpretation Blue Colonies Colonies that appear blue on the agar plate. These colonies contain non-recombinant plasmids, meaning that the plasmid's lacZ gene remains intact and fully functional. As a result, the bacteria express the enzyme β-galactosidase, which cleaves the colorless substrate X-gal, producing a blue-colored product. White Colonies Colonies that remain white on the agar plate. These colonies contain recombinant plasmids, meaning that foreign DNA has been successfully inserted into the MCS, disrupting the lacZ gene. As a result, the bacteria do not produce functional β-galactosidase and cannot cleave X-gal, so no blue color is formed.