Bacterial Transformation Overview

Flashcards covering key concepts and procedures related to bacterial transformation.

What are the five steps of natural transformation in bacteria?

1) Development of competence 2) Binding of DNA 3) Incorporation of DNA 4) Forming a pre-integration complex 5) Integration into the genome of the host cell

What are the essential components of a plasmid for artificial transformation?

A selectable marker, an origin of replication, and a multiple cloning site (MCS).

What role does CaCl2 play in bacterial transformation?

It promotes DNA binding to membranes and helps reorganize lipopolysaccharides and proteins in the outer membrane that influence DNA uptake.

What is the purpose of heat shock during transformation with CaCl2?

It momentarily increases membrane permeability to DNA, allowing for DNA uptake.

How much transformation efficiency is achieved with 10 ng of pUC18 and competent E. coli DH5a cells?

195 CFU/ng, equating to 1.95 X 10^5 CFU/mg DNA.

What is the difference between a spheroplast and a protoplast?

A spheroplast has its cell wall partially removed, while a protoplast has its cell wall entirely removed.

What key factors influence the efficiency of plasmid transformation?

Type of plasmid DNA, state of the host cell, preparation of cells, exposure to DNA, and environmental conditions.

What is the purpose of washing competent cells with MgCl2 after CaCl2 treatment?

To prepare the cells for freezing and improve the competency of the cells for transformation.

Describe the transformation recovery process after the heat shock step.

Add a recovery medium (usually LB) and incubate the cells for 1 hour at 37°C before plating.

What is the transformation efficiency calculation formula?

Transformation Efficiency = CFU / 1 mg DNA.