Describe the role of $\mathrm{CaCl}_2$ in the preparation of competent cells?
$\mathrm{CaCl}_2$ is known to increase the efficiency of DNA uptake to produce transformed bacterial cells. The divalent $\mathrm{Ca}^{+2}$ ions create transient pores on the bacterial cell wall by which the entry of foreign DNA is facilitated into the bacterial cells.
What would happen when one grows a recombinant bacterium in the bioreactor but forget to add antibiotic to the medium in which the recombinant is growing?
In the absence of antibiotic, there will be no pressure on recombinants to retain the plasmid (containing the gene of our interest). Since, maintaining a high copy number of plasmids is a metabolic burden to the microbial cells, it will thus tend to loose the plasmid.
$$ \text { Identify and explain } A, B \text { and } \mathrm{C} \text { in the PCR diagram given below. } $$
$$ \text { Region to be amplified } $$
In PCR, each cycle has three steps
(i) Denaturation of DNA Sample Unwinding of two strand of DNA by heating the sample at $92-94^{\circ} \mathrm{C}$.
(ii) Primer Annealing Primers get positioned on the exposed nucleotides as per base pairing rules.
(iii) Extension of Primers DNA polymerase recognises primers as 'start' tags and begins to extend the primers using the free nucleotides provided in the reaction and the genomic DNA as template.
With each round of reactions, the DNA doubles.
$$ \text { Name the regions marked } A, B \text { and } C \text {. } $$
Region A Bam HI
Region $B$ Pot I
Region C amp $^R$.
E. coli cloning vector pBR322 showing restriction sites (Hind III, Eco RI, Bam HI, Sal I, Pvu II, Pst I, Cla I), Ori and antibiotic resistance genes ( $\mathrm{amp}^R$ and tet ${ }^R$ ).
Rop codes for the proteins involved in the replication of the plasmid.
For selection of recombinants, insertional inactivation of antibiotic marker has been supercoded by insertional inactivation of a marker gene coding for a chromogenic substrate. Give reasons.
In selection of recombinants due to inactivation of antibiotics, the transformed cells are first plated on the antibiotic plate which has not been insertionally inactivated (i.e., ampicillin) and incubated overnight for growth of transformants.
For selection of recombinants, these transformants are replica-plated on second antibiotic (say, tetracycline) plate (which got inactivated due to insertion of gene).
Non-recombinants grow on both the plates (one carrying ampicillin and the other carrying tetracycline) while recombinants will grow only on ampicillin plate. This entire exercise is labourious and takes more time (two overnight incubation) as well.
However, if we choose insertional inactivation of a marker that produces colour in the presence of a chromogenic compound, we can distinguish between the recombinants and non-recombinants on a single medium plate (containing one antibiotic and the chromogenic compound) after overnight growth.