Question

1) You wish to clone a gene into a bacterial plasmid for the production of the E protein to generate antibodies, use in vaccine production, and for biochemical analysis.

A. What expression plasmid will you use and why?

B. What primers to facilitate cloning into this expression vector?

C. What essential features (DNA sequence elements) must the plasmid have to ensure:

i. Plasmid maintenance/amplification in bacteria

ii. Selection of cells transformed with your plasmid

iii. Controlled/inducible expression of Gene E mRNA transcript

iv. Successful translation of the E protein

v. Affinity purification of your E protein

Answer 1

A): "pBR322" with bacterial gene expression can be used as a plasmid.It is 44362bp in length and has two antibiotic resistance gene-Ampicillin resistant(AmpR) protein and Tetracycline resistance(TetR) protein.

It has two genes of special interest; one codes for a protein that enables any host bacterium to resist the lethal effect of antibiotic ampicillin and other confers resistance to tetracycline.

B): One of the primer for Sequencing insert at the Pst1 site of pBR322 is complementary to the "M13" phage vector designed bIa6. This set of Universal primer is used for Rapid sequence determinations of DNA clone into pbr322.

C): pBR322 DNA is commonly used plasmid cloning vector in E coli and also in others.

The molecule is a double standard circle. pBR322 contains the gene for resistance to ampicillin and tetracycline and can be amplified with chloraphenicol.

The molecular weight is 2.53×10⁶ daltons.

1)  High ampicillin rates of pBR322 plasmid DNA is observed in E coli bacteria. The concentration of plasmid per unit of biomass were nearly the same in batch and in fed-batch fermentation of Ecoli-CP79 and Ecoli-CP143.

2)  pBR322 is a commonly used to cloning vector that contains both ampicillin and tetracycline resistance gene as a selectable marker.

E.coli cells transformed with plasmid have a selective advantage in antibiotic treated environment.

3) By gene expression we mean the transcription of a gene into mRNA and its subsequent translation into protein.

Gene expression is primarily controlled at the level of transcription, largely as a result of binding of protein to specific sites on DNA.

The production of the enzyme is controlled by an Operon which consists of related gene on the chromosome with an operator, a promoter, a regulator gene and structural genes.

4) The gene in DNA encode protein molecule which are the workhouse of the cell, carrying out all the function necessary for life.

Expressing a gene means manufacturing its corresponding protein.In translation, mRNA is decoded in the ribosome decoding it to produce specific amino acid chain or polypeptides.

5) Affinity Purification involves a separation of molecules in solution(mobile phase) based on differences in binding interaction with a ligand that is immobilized to stationary material(solid-phase).

Here, the Recombinant protein typically has a tag added, that will help purify it from the rest of proteins found in E.

Diagram of pBR322 vector plasmid: