Selecting recombinants BCMB20005

Flashcards covering key concepts from the lecture on techniques in molecular science, particularly focusing on recombinant plasmid testing and blue/white selection.

What is the role of ampicillin in the agar growth medium?

Ampicillin is an antibiotic that acts as a competitive inhibitor of transpeptidase, necessary for cell wall synthesis.

What gene encodes the enzyme responsible for ampicillin resistance?

The bla gene encodes the enzyme β-lactamase, which hydrolyses the β-lactam ring of ampicillin.

What type of E. coli colonies are considered transformed?

Transformed E. coli colonies containing plasmids appear as white colonies and are ampicillin resistant.

What does blue-white selection involve?

It involves the LacZ gene, where blue colonies indicate non-recombinant plasmids and white colonies indicate recombinant plasmids.

What is X-gal used for in blue-white selection?

X-gal is a chromogenic substrate that is cleaved by β-galactosidase, turning blue for non-recombinant colonies.

What is IPTG and its role in the transformation process?

IPTG is an inducer that removes the lac repressor protein to allow expression of the LacZ gene.

What does it mean if a bacterial colony is white after plating on agar with X-gal and ampicillin?

It indicates a positive clone with a recombinant plasmid that has a disrupted LacZ gene.

What are possible products from the ligation in molecular cloning?

The products include the original plasmid, plasmid with DNA of interest, and digested fragments of genomic DNA.

Which technique can be used to differentiate between DNA fragments ligated into plasmids?

Restriction mapping is used to identify the position of restriction sites and confirm insert orientation.

What are the steps involved in constructing a restriction map?

1. Digest DNA with restriction enzymes 2. Separate digestion products 3. Size digestion products 4. Infer positions of restriction sites 5. Construct a restriction map.

What is the size of the empty plasmid referenced in the lecture?

The empty plasmid has a size of 11 kbp.

What is the significance of having EcoRI and PstI restriction sites?

EcoRI and PstI sites allow for the insertion of foreign DNA and determination of orientation after ligation.

What happens if the LacZ gene is not functional in transformed cells?

No β-galactosidase is produced, resulting in no cleavage of X-gal and no blue product.

How does a recombinant plasmid differ from a non-recombinant plasmid?

A recombinant plasmid contains an inserted DNA fragment that disrupts the LacZ gene, while a non-recombinant does not.

What evidence suggests that a 3-kbp insert has been successfully ligated into a plasmid?

The presence of white colonies on agar plates indicating recombinant plasmids with the inserted DNA fragment.