Blue–white screen

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An LB agar plate showing the result of a blue–white screen.

The blue–white screen is a

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. DNA
of transformation is ligated into a
competent host cell viable for transformation, which are then grown in the presence of X-gal. Cells transformed with vectors containing recombinant DNA
will produce white colonies; cells transformed with non-recombinant plasmids (i.e. only the vector) grow into blue colonies.

Background

bacterial
colony.

The method is based on the principle of α-complementation of the

β-galactosidase was lacking in part of its N-terminus with its residues 11—41 deleted, but it may be complemented by a peptide formed of residues 3—90 of β-galactosidase.[2] M13 filamentous phage containing sequence coding for the first 145 amino acid was later constructed by Messing et al., and α-complementation via the use of a vector was demonstrated by the formation of blue plaques when cells containing the inactive protein were infected by the phage and then grown in plates containing X-gal.[3]

The pUC series of plasmid

cloning vectors by Vieira and Messing was developed from the M13 system and were the first plasmids constructed to take advantage of this screening method.[4] In this method, DNA ligated into the plasmid disrupts the α peptide and therefore the complementation process, and no functional β-galactosidase can form. Cells transformed with plasmid containing an insert therefore form white colonies, while cells transformed with plasmid without an insert form blue colonies; result of a successful ligation can thus be easily identified by the white coloration of cells formed from the unsuccessful blue ones.[5]

Molecular mechanism

A schematic representation of the blue–white assay, used to screen for recombinant vectors

homotetramer
in its active state. However, a mutant β-galactosidase derived from the M15 strain of E. coli has its N-terminal residues 11—41 deleted and this mutant, the ω-peptide, is unable to form a tetramer and is inactive. This mutant form of protein however may return fully to its active tetrameric state in the presence of an N-terminal fragment of the protein, the α-peptide. The rescue of function of the mutant β-galactosidase by the α-peptide is called α-complementation.

In this method of screening, the host E. coli strain carries the lacZ deletion mutant (lacZΔM15) which contains the ω-peptide, while the plasmids used carry the lacZα sequence which encodes the first 59 residues of β-galactosidase, the α-peptide. Neither is functional by itself. However, when the two peptides are expressed together, as when a plasmid containing the lacZα sequence is transformed into a lacZΔM15 cells, they form a functional

β-galactosidase
enzyme.

The blue–white screening method works by disrupting this α-complementation process. The plasmid carries within the lacZα sequence an internal

β-galactosidase
may be formed.

The presence of an active β-galactosidase can be detected by X-gal, a colourless analog of lactose that may be cleaved by β-galactosidase to form 5-bromo-4-chloro-indoxyl, which then spontaneously dimerizes and oxidizes to form a bright blue insoluble pigment 5,5'-dibromo-4,4'-dichloro-indigo. This results in a characteristic blue colour in cells containing a functional β-galactosidase. Blue colonies therefore show that they may contain a vector with an uninterrupted lacZα (therefore no insert), while white colonies, where X-gal is not hydrolyzed, indicate the presence of an insert in lacZα which disrupts the formation of an active β-galactosidase.

The recombinant clones can be further analyzed by isolating and purifying small amounts of plasmid DNA from the transformed colonies and restriction enzymes can be used to cut the clone and determine if it has the fragment of interest.[6] If the DNA is necessary to be sequenced, the plasmids from the colonies will need to be isolated at a point, whether to cut using restriction enzymes or performing other assays.

Practical considerations

The correct type of

competent cells are important considerations when planning a blue–white screen. The plasmid must contain the lacZα, and examples of such plasmids are pUC19 and pBluescript. The E. coli cell should contain the mutant lacZ gene with deleted sequence (i.e. lacZΔM15), and some of the commonly used cells with such genotype are JM109, DH5α, and XL1-Blue. It should also be understood that the lac operon is affected by the presence of glucose. The protein EIIAGlc
, which is involved in glucose import, shuts down lactose permease when glucose is being transported into the cell. The media used in agar plate therefore should not include glucose.

X-gal is light-sensitive and therefore its solution and plates containing X-gal should be stored in the dark. Isopropyl β-D-1-thiogalactopyranoside (IPTG), which functions as the inducer of the lac operon, may be used in the media to enhance the expression of LacZ.

X-gal is an expensive material, thus other methods have been developed in order to screen bacteria. GFP has been developed as an alternative to help screen bacteria. The concept is similar to α-complementation in which a DNA insert can disrupt the coding sequence within a vector and thus disrupt the GFP production resulting in non-fluorescing bacteria.[7] Bacteria that have recombinant vectors (vector + insert), will be white and not express the GFP protein, while non-recombinant (vector), will and fluoresce under UV light. GFP in general has been used as a reporter gene where individuals can definitively determine if a clone carries a gene that researchers are analyzing. On occasion, the medium in which the colonies grow can influence the screen and introduce false-positive results.[8] X-gal on the medium can occasionally degrade to produce a blue color or GFP can lose its fluorescence because of the medium and can impact researchers capabilities to determine colonies with the desire recombinant and those that do not possess it.[9]

Drawbacks

Some white colonies may not contain the desired recombinant plasmid for a number of reasons. The ligated DNA may not be the correct one or not properly ligated, and it is possible for some linearized vector to be transformed, its ends "repaired" and ligated together such that no LacZα is produced and no blue colonies may be formed. Mutation can also lead to the α-fragment not being expressed. A colony with no vector at all will also appear white, and may sometimes appear as satellite colonies after the antibiotic used has been depleted. It is also possible that blue colonies may contain the insert. This occurs when the insert is "in frame" with the LacZα gene and a STOP codon is absent in the insert. This can lead to the expression of a fusion protein that has a functional LacZα if its structure is not disrupted. The correct recombinant construct can sometimes give lighter blue colonies which may complicate its identification.

See also

References

  1. PMID 5339877
    .
  2. PMID 1093175
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  3. PMID 333444
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  4. PMID 6295879
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  5. ISBN 978-0-87969-577-4
    .
  6. OCLC 38325074.{{cite book}}: CS1 maint: multiple names: authors list (link
    )
  7. PMID 23592493
    .
  8. PMID 20459760
    .
  9. PMID 20459760
    .
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