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

4. Using GFP as a biological tracer (this is what GFP is used for in real life applications)
With permission from Marc Zimmer

5. Transformation
Procedure
Overview
Day 1
Day 2

6. 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

7. Bacterial DNA
Bacterial cell
Plasmid DNA
Genomic DNA

8. 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

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

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

11. GFP
Green Fluorescent Protein Ribbon diagram

12. OH OH
a-D-Arabinopyranose OH
f3-D-Arabinopyranose OH
a-D-Arabinofuranose OH
f3-D-Arabinofuranose

13. 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.

14. 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

15. 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

16. 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)

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