pGLO Bacterial Transformation DNA RNA ProteinTrait
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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
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What was the point of this lab?
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What was alive? Escherichia coli, a bacterium Glass E. coli by
Luke Jerram
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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 microscope scanning electron
microscope
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What was the source of new genes? A plasmid, genetically
engineered to carry specific genes
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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
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Bacterial DNA Plasmid DNA Bacterial cell Genomic DNA DNA in
nucleoid region NO nucleus single circular chromosome Bacteria have
a single circular chromosome plasmids Bacteria have accessory DNA,
small circular pieces called plasmids
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pGLO 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
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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.
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Bacterial Transformation Process 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
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Transformation Procedure Suspend bacterial colonies in
Transformation solution Add pGLO plasmid DNA Place tubes on ice
Heat-shock at 42C and place on ice Incubate with nutrient broth
Streak plates: LB LB/amp/+ LB/amp/- LB/amp/ara/+
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Reasons for Performing Each Transformation Step?
1.Transformation solution = CaCI 2 Positive charge of Ca ++ ions
shields negative charge of DNA phosphates Ca ++ O CH 2 O PO O O
Base CH 2 O P O O O Base OH Sugar O Ca ++
What is Nutrient Broth? Luria-Bertani (LB) broth Medium that
contains nutrients for bacterial growth and gene expression
Carbohydrates Amino acids Nucleotides Salts Vitamins
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How do you know which cells transformed (took in plasmid)? pGLO
has reporter genes bla (amp r ) GFP araC
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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!
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ANY QUESTIONS?
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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 cant survive outside lab Not able
to infect animals and plants
STUDENT MANUAL Number of colonies? What is a colony?
Descendants of a single cell!
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STUDENT MANUAL Size of of colonies?
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STUDENT MANUAL Color of colonies?
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STUDENT MANUAL Distribution of colonies?
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STUDENT MANUAL Appearance in UV light?
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STUDENT MANUAL Ability to reproduce in presence of
antibiotic?
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STUDENT MANUAL You should be able to answer questions 1 and
2
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STUDENT MANUAL Lesson 2 page 42 1? You Explain 2? You Explain
3? You Explain 4? You have 2 Control plates You Explain
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STUDENT MANUAL Lesson 3 page 43 1, 2, 3, 4 You do. But first
lets predict!
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PREDICTIONRESULTS Did bacteria get plasmid? Is bacteria
ampicillin resistant? Will bacteria glow in the dark?
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PREDICTIONRESULTS Did bacteria get plasmid? Is bacteria
ampicillin resistant? Will bacteria glow in the dark?
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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?
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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
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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
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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.
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Grow? Glow? Follow protocol On which plates will colonies grow
? Which colonies will glow ?
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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 4200-4500 range.[1][2][3]
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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)
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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 organismsScreenable marker to identify
transgenic organisms
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Using GFP as a biological tracer
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With
permission from Marc Zimmer
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Transformation Procedure Overview Day 1 Day 2
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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
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What is Transformation? Uptake of foreign DNA, often a circular
plasmid GFP Beta-lactamase Ampicillin Resistance
Transcriptional Regulation BAD araC BAD RNA Polymerase Effector
(Arabinose) araC BAD ara Operon RNA Polymerase ZYA ZYA LacI
Effector (Lactose) ZYA LacI lac Operon
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Gene Regulation RNA Polymerase araC ara GFP Operon GFP Gene
araC GFP Gene araC GFP Gene Effector (Arabinose) BAD araC BAD RNA
Polymerase Effector (Arabinose) araC BAD ara Operon
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The Many Faces of Plasmids Scanning electron micrograph of
supercoiled plasmid Graphic representation
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How do you know which cells transformed (took in plasmid)? pGLO
has reporter genes bla (amp r ) 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
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Methods of Transformation Electroporation Electrical shock
makes cell membranes permeable to DNA Calcium Chloride/Heat-Shock
Chemically-competent cells uptake DNA after heat shock