1. transform bacteria with pglo plasmid 3. induce expression of gfp with arabinose
DESCRIPTION
Transformation Lab Creating a GMO by transforming E. coli. 1. Transform bacteria with pGlo plasmid 3. Induce expression of GFP with arabinose. 2. Select for ampicillin resistance. http://www.dnalc.org/harlemdnalab/ BacterialTrans.html. The recombinant pGlo plasmid. - PowerPoint PPT PresentationTRANSCRIPT
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)?