p GLO Bacterial Transformation DNA RNA Protein Trait
p. GLO 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 scanning electron microscope light microscope
What was the source of “new genes”? A plasmid, genetically engineered to carry specific genes
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 “manmade” plasmids are widely used as vectors in molecular cloning
Bacterial DNA in nucleoid region – NO nucleus Bacteria have a single circular chromosome Bacterial cell Bacteria have accessory DNA, small circular pieces called plasmids Plasmid DNA Genomic DNA
p. GLO, p. GLO 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 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.
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) p. GLO plasmids
Transformation Procedure • Suspend bacterial colonies in Transformation solution • Add p. GLO plasmid DNA • Place tubes on ice • Heat-shock at 42°C and place on ice • Incubate with nutrient broth • Streak plates: LB/amp/+ LB/amp/ara/+
Reasons for Performing Each Transformation Step? Ca++ O O P O O CH 2 Base O Sugar 1. Transformation solution = Ca. CI 2 Positive charge of Ca++ ions shields negative charge of DNA phosphates O Ca++ O P O O CH 2 Base O Sugar OH
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)
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)? p. GLO has reporter genes • bla (ampr) • GFP • ara. C
3 Genes of interest in p. GLO 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 ara. C 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 MANUAL Number of colonies? What is a colony? Descendants of a single cell!
STUDENT MANUAL Size of of colonies?
STUDENT MANUAL Color of colonies?
STUDENT MANUAL Distribution of colonies?
STUDENT MANUAL Appearance in UV light?
STUDENT MANUAL Ability to reproduce in presence of antibiotic?
STUDENT MANUAL You 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 Did bacteria get plasmid? Is bacteria ampicillin resistant? Will bacteria glow in the dark? RESULTS
PREDICTION Did bacteria get plasmid? Is bacteria ampicillin resistant? Will bacteria glow in the dark? RESULTS
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 results If successful, state evidence from plates If 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/+p. GLO plate USE THIS AS NUMBER OF COLONIES: 50 PAGE 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 encyclopedia Jump to: navigation, search Left: p. GLO under ambient light Right: p. GLO visualized under ultraviolet light The p. GLO 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 p. GLO plasmid by growing them on +arabinose plates. p. GLO 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) -ara. C, 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 p. GLO plasmid contains an origin (ori), which is a region of the plasmid where replication will originate. The p. GLO plasmid was made famous by researchers in France who used it to produce a green fluorescent rabbit named Alba. Other features on p. GLO, 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. html With 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 • p. GLO plasmid
Transcriptional Regulation ara Operon lac Operon Lac. I Z Y A ara. C Y A B A D RNA Polymerase Z A D Effector (Arabinose) Effector (Lactose) Lac. I B ara. C B A D
Gene Regulation ara GFP Operon ara. C B A D ara. C Effector (Arabinose) ara. C B A D ara. C RNA Polymerase ara. C B A D GFP Gene RNA Polymerase ara. C GFP Gene
The Many Faces of Plasmids Graphic representation Scanning electron micrograph of supercoiled plasmid
How do you know which cells transformed (took in plasmid)? p. GLO 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 • ara. C – 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
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