Question

A researcher has sequenced a gene that has two alleles. she has a mixture of both alleles and clones them into

Answer 1

The blue-white screen refers to a procedure of screening that allows for the brisk and convenient determination of recombinant bacteria in vector-based molecular cloning experiments. In the process, the DNA of interest is ligated into a vector. The blue colonies constitute of self-relegated plasmids that are devoid of DNA inserts obstructing the lac Z gene.  

The white colonies contain bacteria that conduct plasmids, which possess the inserts of DNA fragments that obstruct the lac Z gene. Therefore, the researcher needs to sequence the plasmids of the white colonies prior to knowing which allele it constitutes.  

Answer 2

The researcher can identify colonies containing disease-causing alleles by performing a blue-white screen technique.

Further Explanation:

A technique through which convenient and rapid detection of recombinant bacteria or genes by using molecular cloning experiments that are based on vectors is called a blue-white screen. In this technique, a DNA or allele to be sequenced is inserted into a vector, which is then incorporated into a host cell or E. coli where transformation can take place. These cells then grow in the presence of X-gal. The cells containing the transformed vector with the recombinant DNA will result in white colonies, while the transformed cells containing only the vector will produce blue colonies. The white colonies are the ones containing the gene of interest and can be further selected for sequencing.

In the lac operon, the lacZ gene is responsible for the production of a protein called β-galactosidase. This protein, when present in its active state, exists in the form of a homotetramer. A mutant β-galactosidase lacks certain N-terminal residues and is called ω-peptide, which is not capable of forming a tetramer; therefore, it is inactive. This mutant protein can get back to its active form when the N-terminal containing fragment called α-peptide is present. The restoration of the mutant β-galactosidase function through α-peptide is known as α-complementation.

While performing the screening method, the plasmid contains the α-peptide within which multiple cloning sites (MCS) are present and the E. coli contains the ω-peptide. These MCS can be inserted with a foreign gene through restriction enzyme. This, in turn, disrupts the functionality of the α-peptide or lacZα gene. As a result, the cells comprising the plasmid with the gene of interest does not produce functional β-galactosidase.

Here, when the scientist ligates the plasmid with the specific allele, the lacZα gene becomes non-functional. On insertion of the recombinant plasmid into the E. coli, β-galactosidase is not formed, and no blue colored colonies are produced. Therefore, the cells producing white colonies contain the gene of interest and can be selected for further sequencing.

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Answer Details:

Grade: College Biology

Chapter: Isolation and Analysing Genes

Subject: Biology

Keywords:

Blue-white screen, DNA, genes, alleles, β-galactosidase, α-peptide, ω –peptide, lacZα gene, multiple cloning sites, plasmid, vector, E. coli, transformation, recombinant, non-recombinant, blue colonies, white colonies.