pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP)

What is Transformation? E. coli cell What is Transformation? Uptake of foreign DNA from the external environment

A circular piece of autonomously replicating DNA in bacteria What is a plasmid? A circular piece of autonomously replicating DNA in bacteria May express antibiotic resistance gene or be engineered to express genes of interest pGLO

pGLO contains genes coding for 3 proteins: Beta lactamase (bla) Ampicillin resistance (ampR) Green fluorescent protein (GFP) Aequorea victoria jellyfish gene araC repressor protein Binds to operator & nhibits ara operon transcription

BOOK RETURNS TOMORROW! Prokaryotic Gene Regulation Prokaryotic genes often are bundled into operons. The ara operon contains 3 genes (B,A,D) that code for 3 enzymes needed to break down the sugar arabinose; if arabinose is not present, the ara3 repressor binds to the operator (O) and prevents expression of the genes

Transformation When present, arabinose allosterically binds to the araC protein to enable RNA polymerase to bind to the promoter and transcribe the 3 genes in the arabinose operon, producing the enzymes necessary to digest arabinose Arabinose araC X RNA Pol

Prokaryotic Gene Regulation Is the ara operon inducible or repressible? araC RNA Pol Beta-lactamase

Transformation Regulation GFP has replaced the 3 genes in the arabinose operon, so in the presence of arabinose the GFP gene is expressed, and green fluorescent protein is produced and the bacteria GLOW! RNA Pol GFP

Transformation Procedure Overview Day 2 (cont.) Transformation Procedure Overview Day 1 Day 2 Day 3 10

What is Nutrient Broth? Luria-Bertani (LB) broth Medium that contains nutrients for bacterial growth and gene expression Carbohydrates Amino acids Nucleotides Salts Vitamins

Methods of Transformation Electroporation Electrical shock makes cell membranes permeable to DNA Calcium Chloride/Heat-Shock Chemically-competent cells uptake DNA after heat shock

Transformation E. coli Ca+2 Transformation solution of CaCl2. Ca2+ shields negative charge of DNA phosphates Incubate on ice slows fluid cell membrane 3. Heat-shock Increases permeability of membranes 4. Nutrient broth incubation Allows beta-lactamase expression Ca+2 heat

BOOK RETURNS TOMORROW! Prokaryotic Gene Regulation Prokaryotic genes often are bundled into operons. The ara operon contains 3 genes (B,A,D) that code for 3 enzymes needed to break down the sugar arabinose; if arabinose is not present, the ara3 repressor binds to the operator (O) and prevents expression of the genes

Transformation When present, arabinose allosterically binds to the araC protein to enable RNA polymerase to bind to the promoter and transcribe the 3 genes in the arabinose operon, producing the enzymes necessary to digest arabinose Arabinose araC X RNA Pol

Prokaryotic Gene Regulation Is the ara operon inducible or repressible? araC RNA Pol Beta-lactamase

Transformation Regulation GFP has replaced the 3 genes in the arabinose operon, so in the presence of arabinose the GFP gene is expressed, and green fluorescent protein is produced and the bacteria GLOW! RNA Pol GFP

On which plate(s) will colonies grow? On which plate(s) will colonies GLOW? LB = nutrient broth amp = ampicillin ara = arabinose

Volume Measurement

Questions to consider: Student Inquiry Questions to consider: How important is each step in the lab protocol? What part of the protocol can I manipulate to see a change in the results? Ampicillin concentration Arabinose concentration / timing Heat shock temperature or time Time on ice before and after heat shock Amount of plasmid Amount of bacteria Phase of bacteria used for transformation How do I insure the change I make is what actually affected the outcome? Importance of controlling other variables Collaborative approach / share data Write protocol, get approval, and do it!

More Advanced Questions Student Inquiry More Advanced Questions Are satellite colonies also transformed? What other genes might the pGLO plasmid contain? Can I map the plasmid? Can I remove the pGLO gene? Can I remove the regulation of GFP so I don’t need to add arabinose? Can I detect the presence of the GFP gene using PCR?

Student Inquiry Teacher Considerations What materials and equipment do I have on hand, and what will I need to order? Extra plates, LB, agar, plasmid, ampicillin, arabinose? Incubator, water bath (different temps) Other supplies depending on student questions Consider buying extras in bulk or as refills – many have 1 year + shelf life. What additional prep work will I need? Order supplies Pour plates (different media? different amounts?) Make starter plates (will you need transformed bacteria?) How much time do I want to allow? Limited time? Have students read lab and come up with inquiry questions and protocol before they start. Collaborative approach. Will you need multiple lab periods? Will everyone need the same amount of time?

pGLO Plasmid pGLO Plasmid DNA

Extensions: Green Fluorescent Protein (GFP) Chromatography Kit 1660005EDU GFP Purification Kit Advantages Links to biomanufacturing Biopharmaceutical development Amazing visual results

Extensions: pGLO Kit SDS-PAGE extension 1660013EDU pGLO SDS-PAGE Kit advantages Learn Protein Electrophoresis Visualize GFP on SDS-PAGE gel, it glows still! Extensions: pGLO Kit SDS-PAGE extension 1660013EDU