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1. Transform bacteria with pGlo plasmid 3. Induce expression of GFP with arabinose

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.

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1. Transform bacteria with pGlo plasmid 3. Induce expression of GFP with arabinose

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  1. 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

  2. The recombinant pGlo plasmid araC: a regulator protein needed for transcription of genes araA, -B, and –D in the arabinoseoperon 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, replacingaraA, -B, and -D

  3. Beta lactamase = amoxicillian resistance

  4. The arabinose promotor and BAD operon operon The arabinoseoperon consists of the genes araA, ara B, and araD is adjacent to thepromoter, araC In the presence of arabinose (inducer) araC promotes the expression of the three genes promotor araC/arabinose complex triggers gene expression

  5. 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. Because the GFP gene is inserted adjacent to thearaCpromoter, it is expressed when arabinose is present. promotor new operon promotor activated gene expressed

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

  7. Transformed bacteria take up GFP via the recombined plasmid Recombinant plasmids E. Coli bacteria

  8. Transformation procedure Suspend E. coli colonies in transformation solution 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

  9. 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)?

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