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

Using GFP as a biological tracer (this is what GFP is used for in real life applications) http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer

Transformation Procedure Overview Day 1 Day 2

What is Transformation? Bacteria have normal genomic DNA (shown in pink), plus small circular DNA “plasmids” which contain genes for bacterial self defense. Bacteria can trade these with other bacteria. GFP Uptake of foreign DNA takes place with the circular plasmids. In this lab, we are using specially prepared plasmids that contain the gene for GFP, as well as ampicillin resistance. Beta-lactamase Ampicillin Resistance

Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA

The ‘pGLO’ plasmid we are using contains these genes Beta Lactamase Ampicillin resistance B-lactamase metabolizes any ampicillin present Green Fluorescent Protein (GFP) – Aequorea victoria jellyfish gene araC regulator protein – Regulates GFP transcription ori – the origin site for replication

\'"Cl-· v. Sites of common restriction enzymes , ... (2 ) BspDI ,- c 11a1 (5307 ) SgrAI ( 522 5 ) Afel Nsil ( 9 ) Sites of common restriction enzymes ( 500 4 ) Pflfl · Tthllll (47 48 ) Peil PflMI ( 9 2 0 ) (4339 ) AlwNI Agel (10 74 ) BstEll (1077) Bsml ( l0 97 ) \'"Cl-· v. 3 pGLO 5371 b p 3 -=::::: Nhel ( 13 45 ) Bmtl ( 1349 ) , ji'' (38 96 ) Ps il -- Ncol - Styl (15 10 ) Mlscl ( 1515 ) ... (3 771 ) Dralll PaeR7l · PspXI · Tlil · Xho ,l BstBl ( 1966 ) EooRI (2063) Acc6.51 ( 2 0 7 5 ) Kpnl · Ts pMI · Xmal ( 2 079 ) ' """"'-- Smal ( 2 08 l ) Xbal (209 0 ) Sbfl ( 210 6 ) BfuAI · BspMl ( 2 10 9 ) Sphl (2112) (1765) (3 42 3 ) Ahdl (33 57 ) Bsal (330 5 ) Bgll (3 27 6 ) NmeAIII ( 32 48 ) Asel (3200) Fspl (30 54 ) .,,,, PpuMI · SanDI ( 2286 )

Bacterial Transformation Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids

GFP Green Fluorescent Protein Ribbon diagram

OH OH a-D-Arabinopyranose OH f3-D-Arabinopyranose OH a-D-Arabinofuranose OH f3-D-Arabinofuranose

Methods of Transformation Electroporation Electrical shock makes cell membranes permeable to DNA Calcium Chloride/Heat-Shock Chemically-competent cells uptake DNA after heat shock. This is what we are using.

Transformation Procedure Suspend bacterial colonies in Transformation solution Add pGLO plasmid DNA Place tubes on ice Heat-shock at 42°C and place on ice Incubate with nutrient broth Streak plates

Reasons for Ca++ Performing Each Ca++ O Transformation Step? Ca++ O O P O O CH Each Ca++ O Base Transformation Step? O 2 Sugar 1. Transformation solution = CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates O P O O CH2 Ca++ O Base O Sugar OH

Why Perform Each Transformation Step? Incubate on ice Cell wall GFP Step? Incubate on ice slows fluid cell membrane Heat-shock Increases permeability of membranes 4. Nutrient broth incubation Allows beta-lactamase expression Beta-lactamase (ampicillin resistance)

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