Question

You have isolated a strain of brewer’s yeast (Saccharomyces cerevisiae) in the lab for your second job at a hip new microbrew . You find that this strain ferments more efficiently and adds a superior flavor profile to your brews. You want to clone the strain of yeast and generate a genomic library to determine the genes responsible for this finding. To do so you:

1. Obtain fragments of the whole yeast genome (DNA) through restriction digestion
2. Open your vector and ligate the DNA fragments
3. Transform E. coli with these vectors
4. Select for E. coli that have obtained vectors
5. Screen for E. coli transformed with recombinant plasmids
6. Obtain recombinant plasmids from the library and transform yeast with them
7. Clone by complementation the gene that can give superior fermentation.

Note: the cloning site is within the lacZ gene.

a) (3 points) In step 3 you transform E.coli with your plasmids and plate them on a solid agar medium to grow and isolate colonies. What medium should you used to distinguish the bacteria transformed with recombinant vectors from ones that carry non-recombinant vectors? Briefly explain how the medium allows for you to distinguish. (hint: look up the substrate X-gal)

b) (2 points) You identify a recombinant vector that passes your complementation test. You are curious whether this vector will cause bacteria to ferment sugars for brewing. Provide and explain two reasons why this recombinant vector will NOT work in bacteria.

Answer 1

a) Transformed E.coli vectors will be selected by using antibiotic resistant markers for example AmpR (Ampicillin resistance) present in the plasmid. The E. Coli that are transformed by the vector plasmid will survive on the media containing ampicillin. The blue white screening is an efficient technique to rapidly identify the recombinant and non recombinant strain. Briefly, the cloning site is present in LacZ gene which produces β-galactoisdase enzyme that metabolizes the lactose. If the recombinant DNA is integrated at the cloning site LacZ gene will be disrupted and because of that β-galactoisdase enzyme will not produce. Whereas, non-recombinants will produce functional β-galactoisdase enzyme. For screening instead of lactose a chromogenic analog X-gal is used . The X-gal is hydrolyzed by β-galactoisdase enzyme to form 5-bromo-4-chloro-indoxyl, which spontaneously dimerizes to produce an insoluble blue pigment called 5,5’-dibromo-4,4’-dichloro-indigo. Similarly, Isopropyl β-D-1-thiogalactopyranoside (IPTG) a non-metabolizing analog of galactose is used as an inducer of LacZ operon. Therefore, X gal and IPTG is used in media for screening.

b) The two major reason that the recombinant vector will not work in bacteria is

1) In bacteria transcription is coupled with translation, whereas, eukaryotic transcript undergoes post transcriptional modifications like mRNA splicing.

2) The eukaryotic proteins after translation undergoes a process called as post translational modification, for example glycosylation, which required for its optimal activity.