What is the blue white screening method?
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.
Can you do blue white screening without IPTG?
In some blue/white screening systems, an additional reagent must be used: IPTG (isopropylthiogalactoside). IPTG is an inducer that de-represses lacZ expression (it turns the gene on). In some cases, without IPTG, not enough β-galactosidase is produced to turn the colony blue even if the lacZ gene is intact.
What is the purpose of screening in gene cloning?
A screen will help you more easily identify successful clones so you have to weed through fewer colonies after your experiments. As a common example, a selection will leave you with the colonies that contain your plasmid backbone, as it often relies on antibiotic resistance.
What is the purpose of blue white screening?
The blue–white screen is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in vector-based molecular cloning experiments. This method of screening is usually performed using a suitable bacterial strain, but other organisms such as yeast may also be used.
What is the function of lacZ?
The LacZ protein codes for an enzyme called β-galactosidase, which is an essential part of the metabolism of lactose. It cleaves (separates) a single disaccharide lactose molecule into far more digestible glucose and galactose.
What is blue white screening Slideshare?
I) BLUE-WHITE SCREENING The use of chromogenic substrate to detect a particular enzymatic activity is the basis to screen the desired clone. The colourless compound X-gal or 5-bromo-4-chloro-3-indolyl-β-D-galactoside used in this screening method is a substrate for β-galactosidase.
What is the function of ampicillin in blue white screening?
Using Ampicillin (or other appropriate antibiotic) in your growth medium should prevent bacteria that did not take up the plasmid during the transformation from growing. This way you can be fairly confident that the white colonies you see on your screening plate contain plasmid with insert.
What is lacZ and why it is important?
The LacZ protein codes for an enzyme called β-galactosidase, which is an essential part of the metabolism of lactose. The enzyme also has the ability to catalyze the conversion of lactose to allolactose, or to attach allolactose to monosaccharides.
What is lacZ gene?
The lacZ gene encodes β-galactosidase, which degrades lactose. The lacY gene product, lactose permease, transports lactose into the cell, and the lacA gene product, lactose acetylase, has an unknown and not usually necessary function. The lac operon is repressed by LacI, encoded by lacI.
How is the blue white screening method employed?
The blue/white screening method relies on the principle of α-complementation of the β-galactosidase gene, where a fragment of the lacZ gene (lacZα) in the plasmid can complement another mutant lacZ gene (lacZΔM15) in the bacterial cell. Each of these genes produces a non-functional peptide.
How is blue-white screening used for colony selection?
Blue-White Screening & Protocols for Colony Selection 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.
How is blue white screening used to select plasmids?
Although blue-white screening is probably the most widespread way to select for plasmids containing an insert, there are other methods. Positive selection vectors encode a gene which, when expressed, is lethal to the cell. Cloning fragments are inserted into an MCS in the center of this gene, disrupting the lethality.
What are the steps in blue white screening?
The complete protocol of blue-white screening includes 3 important steps: Ligation: ligation of foreign DNA into MCS of the plasmid vector Transformation: introduction of plasmid vector with foreign DNA insert into competent E. coli Screening: blue-white screening to identify recombinant bacterial colonies
How does blue white screening work in E coli?
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. The presence of lactose in the surrounding environment triggers the lacZ operon in E. coli.