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    Blue-White Colony Screening

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    Shalizma Khandaker Orni
    Blue-white colony screening uses the enzyme β-galactosidase, encoded by the lacZ gene in E. coli, to detect recombinant bacteria. β-galactosidase metabolizes X-Gal to form a blue pigment. Bacteria containing plasmids without inserts of foreign DNA form blue colonies, while bacteria with inserts that disrupt the lacZ gene form white colonies lacking β-galactosidase activity. This allows identification of recombinant colonies containing the desired inserted DNA. However, some limitations include false positives from non-functional lacZ genes or small in-frame inserts causing ambiguous light blue colonies.

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    Blue-White Colony Screening

    Uploaded by

    Shalizma Khandaker Orni
    71 views1 page
    Blue-white colony screening uses the enzyme β-galactosidase, encoded by the lacZ gene in E. coli, to detect recombinant bacteria. β-galactosidase metabolizes X-Gal to form a blue pigment. Bacteria containing plasmids without inserts of foreign DNA form blue colonies, while bacteria with inserts that disrupt the lacZ gene form white colonies lacking β-galactosidase activity. This allows identification of recombinant colonies containing the desired inserted DNA. However, some limitations include false positives from non-functional lacZ genes or small in-frame inserts causing ambiguous light blue colonies.

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    Blue-white colony screening uses the enzyme β-galactosidase, encoded by the lacZ gene in E. coli, to detect recombinant bacteria. β-galactosidase metabolizes X-Gal to form a blue pigment. Bacteria containing plasmids without inserts of foreign DNA form blue colonies, while bacteria with inserts that disrupt the lacZ gene form white colonies lacking β-galactosidase activity. This allows identification of recombinant colonies containing the desired inserted DNA. However, some limitations include false positives from non-functional lacZ genes or small in-frame inserts causing ambiguous light blue colonies.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    71 views1 page

    Blue-White Colony Screening

    Uploaded by

    Shalizma Khandaker Orni
    Blue-white colony screening uses the enzyme β-galactosidase, encoded by the lacZ gene in E. coli, to detect recombinant bacteria. β-galactosidase metabolizes X-Gal to form a blue pigment. Bacteria containing plasmids without inserts of foreign DNA form blue colonies, while bacteria with inserts that disrupt the lacZ gene form white colonies lacking β-galactosidase activity. This allows identification of recombinant colonies containing the desired inserted DNA. However, some limitations include false positives from non-functional lacZ genes or small in-frame inserts causing ambiguous light blue colonies.

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    Blue-White Colony Screening

    Introduction

    Blue-white screening of bacterial colonies is a common and effective molecular biology tool used to
    detect recombinant bacteria in genetic cloning experiments. The enzymatic activity of β-galactosidase, a
    tetrameric enzyme encoded by the lacZ α gene found in E. coli is essential to this technique because it
    metabolizes lactose to form glucose and galactose. Additionally, β-galactosidase hydrolyzes another
    substrate, X-Gal, resulting in 5-bromo-4-chloro-indoxyl, which dimerizes to form a blue pigment.

    How does it work?

    The α-complementation mechanism has allowed β-galactosidase to be a promising method for


    molecular cloning. In α-complementation, the deletion of a particular fragment of the lacZ ω gene in
    bacteria resulting in inactive β-galactosidase is overcome by a plasmid containing the deleted fragment.
    The plasmids used in cloning commonly contain a portion of the lacZ α gene, while the E. coli host strain
    has a lacZ ω deletion mutation. Thus, functional β-galactosidase is generated during transformation
    when bacteria that carry the deletion take up the plasmid containing the deleted lacZ α fragment.
    Nonetheless, if the plasmid taken up by the bacteria is carrying a piece of DNA (DNA of interest ligated
    to the plasmid using restriction sites during the cloning process) that disrupts the segment of the lacZ α
    gene, recombinant bacteria result. Then, there can be no alpha supplementation, so no functional β-
    galactosidase is produced.

    To perform blue-white colony screening after transformation, X-Gal is added alongside with Isopropyl β-
    D1-thiogalactopyranoside (IPTG), an inducer of lacZ ω gene expression. The blue colonies formed have
    bacteria containing functional β-galactosidase, indicating that the plasmid which was taken up during
    transformation did not contain the DNA of interest. The white colonies cannot metabolize X-Gal to
    produce the blue color, because they do not produce functional β-galactosidase after taking up plasmid
    containing the inserted DNA and disrupting the lacZ α gene. These white colonies contain the
    recombinant bacteria and should be selected.

    Limitations of blue-white screening

     The blue-white technique is only a screening procedure; it is not a selection technique.


     The lacZ gene in the vector may sometimes be non-functional and may not produce β-
    galactosidase. The resulting colony will not be recombinant but will appear white.
     Even if a small sequence of foreign DNA may be inserted into MCS and change the reading
    frame of lacZ gene. This results in false positive white colonies.
     Small inserts within the reading frame of lacZ may produce ambiguous light blue colonies as β-
    galactosidase is only partially inactivated.

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