Question

A lab that involves transformation and expression of ampicillin resistant genes.

a. Outline the steps in transformation.

b.Outline the steps involved in expressing new genes starting with the gene and ending with an ampicillin resistant E. coli colony

Answer 1

a) Transformation of the ampicillin resistant gene:

1. First, you have to make competent cells. For this, you have to grow the E coli cells up to mid-log phase.

2. Then centrifuge the culture medium and collect the cell pellet. Then dissolve the cell palate in CaCl₂ solution. Now you got your competent cells.

3. Now you have to do the transformation step. For this, you take some volume of ampicillin resistant plasmid and some volume of competent cell in a microcentrifuge tube and give a heat shock for 1 min or you can do electroporation (1 or 2 pulses) also. Now the plasmids will get inside the cells.

4. Then after the transformation, you have to revive the cells by adding 1 ml of broth solution in those microcentrifuge tubes and growing in inside the incubator for 1 hr.

b) Expression of the ampicillin resistant gene:

Generally, most of the genes are under the regulatory sequence of lac - operon. So, here also, the ampicillin resistant gene is under the control of the lac-operon regulatory sequence.

1) First, you have to grow the cells to mid-log phase (you can identify it by measuring the OD or absorbance of the culture medium).

2. Then you have to induce the gene expression of the culture cells by adding IPTG. It acts as an analog of allolactose and will release the lac repressor from the promoter of the ampicillin gene and as a result, the ampicillin gene will start expressing.