Bacterial Transformation

RET Summer 2007

Overall Picture

Bio-Rad pGLO Transformation

Insertion of GFP gene into HB101 E. coli

Transformation

The process of transferring foreign DNA

fragments into a recipient (host) cell for

growth and replication

Our host cells: HB101 E. coli

Our foreign DNA: GFP & -lactamase

genes (contained in the pGLO plasmid)

Plasmids

Plasmids

small (1-1000 kb)

circular

extrachromosomal DNA

Growth is independent of the hosts cell cycle;

amplification of gene product

A type of cloning vector used to carry a gene not

found in the bacterial hosts chromosome

Overall Transformation

Process

1. The plasmid vector must be cut with a

restriction endonuclease (aka: restriction

enzyme)

2. DNA ligase joins the DNA fragment & vector

DNA

3. Host cell is made competent so can plasmid can

enter

4. Transformed cells are grown on selection media

Overall Transformation

Process

1. The plasmid vector must be cut with a

restriction endonuclease (aka: restriction

enzyme)

2. DNA ligase joins the DNA fragment & vector

DNA

3. Host cell is made competent so can plasmid can

enter

4. Transformed cells are grown on selection media

Restriction Enzymes Endonucleases:

in nature, they protect bacteria

from intruding DNA

cut up (restrict) the viral DNA

cut only at very specific

nucleotide sequences

Restriction site:

recognition sequence for a

particular restriction enzyme

Restriction fragments:

segments of DNA cut by

restriction enzymes in a

reproducible way

DNA ligase:

joins the sticky ends of DNA

fragments

Overall Transformation

Process

1. The plasmid vector must be cut with a

restriction endonuclease (aka: restriction

enzyme)

2. DNA ligase joins the DNA fragment & vector

DNA

3. Host cell is made competent so can plasmid can

enter

4. Transformed cells are grown on selection media

Transformation of Bacteria

Generally occurs through heat shock and

addition of a divalent cation to permeabilize

the membrane

Competent cells are those capable of taking up

the plasmid

Overall Transformation

Process

1. The plasmid vector must be cut with a

restriction endonuclease (aka: restriction

enzyme)

2. DNA ligase joins the DNA fragment & vector

DNA

3. Host cell is made competent so can plasmid can

enter

4. Transformed cells are grown on selection media

Selection

A selective medium is used to determine which

bacterial cells contain the antibiotic resistant

plasmid insert and which do not

For example, a bacterium containing a plasmid

with resistance to a particular antibiotic

(ampicillin) will grow on medium that contains

that antibiotic

In addition, our plasmid contains a regulatory

element that activates the GFP gene only in the

presence of arabinose

Selection Media

LB plates:

LB + amp:

LB + amp + ara:

Control (-pGLO)

Should contain only cells with the amp-

resistant pGLO plasmid; colonies appear

white (-pGLO, + pGLO)

Should contain only cells with the

amp-resistant pGLO plasmid;

colonies floresce green (+pGLO)

Factors that Affect Yield and

Quality of Plasmid DNA

Plasmid copy number

Host strain used, carbohydrate production

Culture medium, selection, and culture time

Want to harvest during log growth phase

Transformation Applications

GFP Uses

Use as a reporter molecule to

follow changes in gene

expression over time

Nondestructive, nontoxic

Coding sequence can be

cloned into a variety of

vectors

GFP keeps its fluorescence in

cells from different species

Can be tracked in living cells

over to time to study

development

Can be directed to specific subcellular compartments

Can combine GFP coding region with the regulatory region for another gene and observe changes in gene expression

Can be used to make a fusion protein to study localization, turnover & intracellular associations of native protein

GFP gene is switched on when cells are grown in the presence of arabinose