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DNA. RNA. Protein. Trait. pGLO Bacterial Transformation. SAFETY FIRST!. 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. What was the point of this lab?. What was alive?. - PowerPoint PPT Presentation
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pGLO Bacterial Transformation
DNA RNA Protein Trait
SAFETY FIRST!
• 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
What was the point of this lab?
What was alive?
Escherichia coli, a bacterium
Glass E. coli by Luke Jerram
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
light microscopescanning electron microscope
What was the source of “new genes”?
A plasmid, genetically engineered to carry specific genes
Symbol Bacterial structure Illustration of E. coli K-12
Plasmid containing a few genes
Circular bacterial chromosome
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
Bacterial DNA
Plasmid DNA
Bacterial cell
Genomic DNA
DNA in nucleoid region – NO nucleus
Bacteria have a single circular chromosome
Bacteria have accessory DNA, small circular pieces called plasmids
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
How did you get the plasmid intothe 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.
Bacterial TransformationProcess by which bacteria take up plasmids from the environment and express the plasmid genes
Beta lactamase(ampicillin resistance)
pGLO plasmids
Bacterial chromosomal DNA
Cell wall
GFP
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/+
Reasons for Performing Each Transformation Step?
1. Transformation solution = CaCI2Positive charge of Ca++ ions shields negative charge of DNA phosphates
Ca++
Ca++
OCH2
O
P OO
O Base
CH2
O
PO
O
O
Base
OH
Sugar
Sugar
OCa++
Why Perform Each Transformation Step?
2. Incubate on iceslows fluid cell membrane
3. Heat-shockIncreases permeability of membranes
4. Nutrient broth incubationAllows beta-lactamase expression
Beta-lactamase(ampicillin resistance)
Cell wall
GFP
What is Nutrient Broth?
• Luria-Bertani (LB) broth• Medium that contains nutrients for bacterial
growth and gene expression– Carbohydrates– Amino acids– Nucleotides– Salts– Vitamins
How do you know which cells transformed (took in plasmid)?
pGLO has reporter genes• bla (ampr) • GFP• araC
3 Genes of interest in pGLO plasmid • 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”
This is DNA!
ANY QUESTIONS?
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
STUDENT MANUAL
• Lesson 1 page 34• Phenotype – physical appearance• Starter Plate:
Let’s answer the questions together.
STUDENT MANUALNumber of colonies?
What is a colony?Descendants of a single cell!
STUDENT MANUALSize of of colonies?
STUDENT MANUALColor of colonies?
STUDENT MANUALDistribution of colonies?
STUDENT MANUALAppearance in UV light?
STUDENT MANUALAbility to reproduce in presence of antibiotic?
STUDENT MANUALYou should be able to answer questions 1 and 2
STUDENT MANUAL
• Lesson 2 page 42• 1? You Explain• 2? You Explain• 3? You Explain• 4? You have 2 Control plates – You Explain
STUDENT MANUAL
• Lesson 3 page 43• 1, 2, 3, 4 You do.• But first let’s predict!
PREDICTION RESULTS
Did bacteria get plasmid?Is bacteria ampicillin resistant?Will bacteria glow in the dark?
PREDICTION RESULTS
Did bacteria get plasmid?Is bacteria ampicillin resistant?Will bacteria glow in the dark?
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?
STUDENT MANUAL
• Lesson 3 page 45• 1 The sample did not flouresce• 2, 3 You do• 4 You must answer based on you resultsIf successful, state evidence from platesIf unsuccessful, state evidence from plates
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
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: 50PAGE 51: REPORT TO BISHOP AS SOON AS YOU HAVE NUMBERS. WE WILL SHARE NUMBERS LATER.
Grow? Glow?
• Follow protocol
• On which plates will colonies grow?
• Which colonies will glow?
From Wikipedia, the free encyclopediaJump to: navigation, search Left: pGLO under ambient light
Right: pGLO visualized under ultraviolet lightThe 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 4200-4500 range.[1][2][3]
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)
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
Using GFP as a biological tracer
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.htmlWith permission from Marc Zimmer
Transformation Procedure Overview
Day 1
Day 2
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
What is Transformation? • Uptake of foreign
DNA, often a circular plasmid
GFP
Beta-lactamase
Ampicillin Resistance
Transcriptional Regulation
• Lactose operon
• Arabinose operon• pGLO plasmid
Transcriptional Regulation
B A DaraC
B A DaraC
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
RNA PolymeraseZ Y A
Z Y ALacI
Effector (Lactose)
Z Y ALacI
lac Operon
Gene Regulation
RNA Polymerase
araC
ara GFP OperonGFP Gene
araC GFP Gene
araC GFP Gene
Effector (Arabinose)
B A DaraC
B A DaraC
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
The Many Faces of Plasmids
Scanning electron micrograph of supercoiled plasmid
Graphic representation
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
Methods of Transformation
• Electroporation– Electrical shock makes cell membranes
permeable to DNA
• Calcium Chloride/Heat-Shock– Chemically-competent cells uptake DNA after
heat shock