1. pGLO Bacterial Transformation
DNA
RNA
Protein
Trait

2. SAFETY FIRST! JUMP TO SLIDE 29 WEAR GLOVES DO NOT OPEN PLATES
CLOROX LAB STATION WHEN DONE
BRING PLATES TO BISHOP TO BE DESTROYED.
WASH HANDS WITH SOAP – HAPPY BIRTHDAY
JUMP TO SLIDE 29

3. What was the point of this lab?

4. Escherichia coli, a bacterium
What was alive?
Escherichia coli, a bacterium
Glass E. coli by Luke Jerram

5. Escherichia coli (E. coli)
Found in human large intestines
Model organism for molecular biology
model organism – species that has been extensively studied to better understand biological phenomena and give insight to workings of other organisms
“White rat” of molecular biology
scanning electron microscope
light microscope

6. What was the source of “new genes”?
A plasmid, genetically engineered to carry specific genes

7. Plasmid
commonly found as small circular, double-stranded DNA molecules in bacteria; a NORMAL component of prokaryotes (and a few eukaryotes!)
physically separate from, and replicate independently of, chromosomal DNA within a cell
carry genes that may benefit survival of the organism (e.g. antibiotic resistance)
may be modified to express proteins of interest; these “man-made” plasmids are widely used as vectors in molecular cloning

8. Bacterial DNA DNA in nucleoid region – NO nucleus
Bacteria have a single circular chromosome
Bacteria have accessory DNA, small circular pieces called plasmids
Bacterial cell
Plasmid DNA
Genomic DNA

9. pGLO, a recombinant plasmid & vector
Recombinant plasmid – plasmid into which DNA fragments or genes have been inserted
Vector – plasmid used experimentally as tool to clone, transfer, and manipulate genes
Because bacteria divide rapidly, they can be used as “factories” to copy DNA fragments in large quantities

10. How did you get the plasmid into the E. coli?
TRANSFORMATION - Process by which bacteria take up plasmids from the environment and express the plasmid genes EXPRESS – make proteins
TRANSFORMATION IS A NATURAL PROCESS BUT SOME BACTERIA ARE “BETTER” AT IT THAN OTHERS. Our procedure was developed to improve the likelihood E. coli will transform.

11. Bacterial Transformation Process by which bacteria take up plasmids from the environment and express the plasmid genes
Cell wall
GFP
Bacterial
chromosomal DNA
Beta lactamase
(ampicillin resistance)
pGLO plasmids

12. 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: LB
LB/amp/+
LB/amp/-
LB/amp/ara/+

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

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

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

16. How do you know which cells transformed (took in plasmid)?
pGLO has reporter genes
bla (ampr)
GFP
araC

17. 3 Genes of interest in pGLO plasmid
This is DNA!
Gene for Beta Lactamase (bla)
Protein that inactivates the antibiotic ampicillin
Gene for Green Fluorescent Protein (GFP)
Aequorea victoria jellyfish gene
Protein fluoresces green after absorbing UV or blue light
Gene for araC regulator protein
Protein that regulates GFP transcription – “turns GFP gene on”

18. ANY QUESTIONS?

19. STUDENT MANUAL Lesson 1 page 33 1, 2, 4 – you should be able to answer
3 safety ideas?
Does not produce toxic compounds which make people sick
Grows well in lab but can’t survive outside lab
Not able to infect animals and plants

20. STUDENT MANUAL Lesson 1 page 34 Phenotype – physical appearance
Starter Plate:
Let’s answer the questions together.

21. STUDENT MANUAL What is a colony? Descendants of a single cell!
Number of colonies?
What is a colony?
Descendants of a single cell!

22. STUDENT MANUAL
Size of of colonies?

23. STUDENT MANUAL
Color of colonies?

24. STUDENT MANUAL
Distribution of colonies?

25. STUDENT MANUAL
Appearance in UV light?

26. STUDENT MANUAL
Ability to reproduce in presence of antibiotic?

27. STUDENT MANUAL
You should be able to answer questions 1 and 2

28. STUDENT MANUAL Lesson 2 page 42 1? You Explain 2? You Explain
4? You have 2 Control plates – You Explain

29. STUDENT MANUAL Lesson 3 page 43 1, 2, 3, 4 You do.
But first let’s predict!

30. PREDICTION RESULTS Did bacteria get plasmid?
Is bacteria ampicillin resistant?
Will bacteria glow in the dark?

31. PREDICTION RESULTS Did bacteria get plasmid?
Is bacteria ampicillin resistant?
Will bacteria glow in the dark?

33. STUDENT MANUAL Lesson 3 page 44 1, 2 You do
3 Badly worded. Infer cells are expressing the bla gene – making a protein that inactivates ampicillin
4 Hint what plates should you compare and why?

34. STUDENT MANUAL Lesson 3 page 45 1 The sample did not flouresce
2, 3 You do
4 You must answer based on you results
If successful, state evidence from plates
If unsuccessful, state evidence from plates

35. STUDENT MANUAL Lesson 3 page 46 Yes, E. coli grow on the LB plate
1, 2 you answer
3 a. What are the 2 factors?
3 b. What does arabinose do? What does UV light do
3 c. You answer

36. STUDENT MANUAL Lesson 4 follow the instructions and do the math.
Page 48 #1. Some of you have no colonies on the LB/amp/ara/+pGLO plate
USE THIS AS NUMBER OF COLONIES: 50
PAGE 51: REPORT TO BISHOP AS SOON AS YOU HAVE NUMBERS. WE WILL SHARE NUMBERS LATER.

38. Grow? Glow? Follow protocol On which plates will colonies grow?
LB/Amp
Follow protocol
On which plates will colonies grow?
Which colonies will glow?
LB/Amp/Ara LB

39. From Wikipedia, the free encyclopedia
Jump to: navigation, search
Left: pGLO under ambient light
Right: pGLO visualized under ultraviolet light
The pGLO plasmid is an engineered plasmid used in biotechnology as a vector for creating genetically modified organisms. The plasmid contains several reporter genes, most notably for the green fluorescent protein (GFP) and the ampicillin resistance gene. GFP was isolated from the jelly fish Aequorea victoria. Because it shares a bidirectional promoter with a gene for metabolizing arabinose, the GFP gene is expressed in the presence of arabinose, which makes the transgenic organism fluoresce under UV light. GFP can be induced in bacteria containing the pGLO plasmid by growing them on +arabinose plates.
pGLO is made up of three genes that are joined together using recombinant DNA technology. They are as follows:
-Bla, which codes for the enzyme beta-lactamase giving the transformed bacteria resistance to the beta-lactam family of antibiotics (such as of the penicillin family)
-araC, a promoter region that regulates the expression of GFP (specifically, the GFP gene will be expressed only in the presence of arabinose)
-GFP, the green fluorescent protein
Like most other circular plasmids, the pGLO plasmid contains an origin (ori), which is a region of the plasmid where replication will originate. The pGLO plasmid was made famous by researchers in France who used it to produce a green fluorescent rabbit named Alba.
Other features on pGLO, like most other plasmids, include: a selectable marker, Ori (origin of replication), and an MCS (multiple cloning site) located at the end of the GFP gene. The plasmid is 5371 base pairs long. In supercoiled form, it runs on an agarose gel in the range.[1][2][3]

40.                                              
The green fluorescent protein GFP consists of 238 amino acids, linked together in a long chain. This chain folds up into the shape of a beer can. Inside the beer can structure the amino acids 65, 66 and 67 form the chemical group that absorbs UV and blue light, and fluoresces green. (Credit: Image courtesy of Nobel Foundation)

42. Links to Real-world GFP is a visual marker
Study of biological processes (example: synthesis of proteins)
Localization and regulation of gene expression
Cell movement
Cell fate during development
Formation of different organs
Screenable marker to identify transgenic organisms

44. Using GFP as a biological tracer
With permission from Marc Zimmer

45. Transformation Procedure Overview
Day 1
Day 2

46. What is a plasmid? A circular piece of autonomously replicating DNA
Originally evolved by bacteria
May express antibiotic resistance gene
or be modified to express proteins of interest

47. What is Transformation?
GFP
Uptake of foreign DNA, often a circular plasmid
Beta-lactamase
Ampicillin
Resistance

48. Transcriptional Regulation
Lactose operon
Arabinose operon
pGLO plasmid

49. Transcriptional Regulation
RNA Polymerase Z Y A
LacI
Effector (Lactose)
lac Operon B A D
araC
RNA Polymerase
Effector (Arabinose)
ara Operon

50. Gene Regulation B A D ara Operon ara GFP Operon GFP Gene
araC
RNA Polymerase
Effector (Arabinose)
ara Operon
RNA Polymerase
araC
ara GFP Operon
GFP Gene
Effector (Arabinose)

51. The Many Faces of Plasmids
Graphic representation
Scanning electron micrograph of supercoiled plasmid

52. How do you know which cells transformed (took in plasmid)?
pGLO has reporter genes
bla (ampr) – transformed cells will grow on agar containing ampicillin, an antibiotic
Codes for enzyme called beta lactamase
GFP – transformed cells will glow in UV light
Codes for green fluorescent protein
araC
regulates expression of GFP; GFP expressed only in presences of arabinose, a sugar

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