Transcript pGlo
Transformation Lab Creating a GMO by transforming E. coli 1. Transform bacteria with pGlo plasmid 2. Select for ampicillin resistance 3. Induce expression of GFP with arabinose http://www.dnalc.org/harlemdnala b/BacterialTrans.html The recombinant pGlo plasmid araC: a regulator protein needed for transcription of genes araA, -B, and –D in the arabinose operon ori: Plasmid replication origin bla: Beta-lactamase an enzyme responsible for bacterial resistance to antibiotics including amoxicillin gfp: gene for green florescent protein from A. victoria, replacing araA, -B, and -D Beta lactamase = amoxicillian resistance The arabinose promotor and BAD operon operon 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 promotor araC/arabinose complex triggers gene expression The arabinose promotor and pglo operon In the pGlo plasmid, the BAD operon has been replaced with the GFP gene. This protein glows under UV light. promotor new operon promotor activated Because the GFP gene is inserted adjacent to the araC promoter, it is expressed when arabinose is present. gene expressed Aequorea victoria, original source of the green fluorescent protein (GFP) Transformed bacteria take up GFP via the recombined plasmid Recombinant plasmids E. Coli bacteria 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 42C Increases 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)?