1. Transform bacteria with pGlo plasmid

3. Induce expression of GFP with arabinose

Transformation LabCreating a GMO by transforming E. coli

2. Select for ampicillin resistance

http://www.dnalc.org/harlemdnalab/BacterialTrans.html

bla: Beta-lactamase an enzyme responsible for bacterial resistance to antibiotics including amoxicillin

ori: Plasmid replication origin

The recombinant pGlo plasmidaraC: a regulator protein needed for transcription of genes araA, -B, and –D in the arabinose operon

gfp: gene for green florescent protein from A. victoria, replacing araA, -B, and -D

Beta lactamase = amoxicillian resistance

The arabinose operon consists of the genes ara A, ara B, and ara D is adjacent to the promoter, araC

In the presence of arabinose (inducer) araC promotes the expression of the three genes

The arabinose promotor and BAD operon

promotor

operon

araC/arabinose complex triggers gene expression

In the pGlo plasmid, the BAD operon has been replaced with the GFP gene. This protein glows under UV light.

Because the GFP gene is inserted adjacent to the araC promoter, it is expressed when arabinose is present.

The arabinose promotor and pglo operon

gene expressed

promotor new operon

promotor activated

Aequorea victoria, original source of the green fluorescent protein (GFP)

E. Coli bacteria

Recombinant plasmids

Transformed bacteria take up GFP via the recombined plasmid

Transformation procedure

1. Suspend E. coli colonies in transformation solution

2. Add loop of pGLO plasmid to ‘+’ tube

3. Incubate tubes on ice 10 minutes slows fluid cell membranes

4. Heat shock 50 seconds at 42CIncreases membrane permeability

5. Ice again, 2 minutes

6. Incubate 10 min with LB broth allows blac expression (antibiotic resistance)

7. Streak the plate; incubate

add a bacterial colony to each tube

add pglo to ‘+’ tube

‘-’ tube lacks plasmid

Hypothesize: which will grow bacteria? Which will grow only ampicillin-resistant colonies (selection)? Which will glow in UV light (GFP expression)?