pGLO Bacterial pGLO Bacterial Transformation, Transformation,

Purification and SDS Purification and SDS gelgel

Timeline Timeline TranformationTranformation

Monday1/7 Lecture and discussion

Tuesday 1/8 Transformation LaboratoryTransform cells and spread plates

Wednesday 1/9 (FIRE)Data Collection and AnalysisObserve transformants and controlsAnalyze and interpret resultsInoculationPicking Colonies and Inoculating Cell Cultures

HW Extension ActivitiesCalculate transformation efficiency

Timeline PurificationTimeline PurificationThursday 1/10 Purification Phase 1

Bacterial Concentration and Lysis

Tuesday 1/15 Purification Phase 2Removing Bacterial Debris

Purification Phase 3

Protein Chromatography

Timeline SDS GelTimeline SDS GelThursday 1/17—Prepare Samples, Run gel, stain,

destain (helper stay 7th)Friday 1/18—View Results, turn in labMonday 1/21—post lab quiz, turn in lab

Prep for Prep for Transformation LabTransformation Lab

• 3-7 days prior to labo Prepare agar plates, arabinose and ampicillan,(page 13-15)

o Aliquot solutions (page 17)

• 24-36 hr prior (page 16-17)o Rehydrate E. coli o Streak starter plateso Prepare pGLO plasmid

Prep for Lab Prep for Lab PurificationPurification

• Before Inoculation Lab (Page 6-7)o Prepare liquid culture media, shaking incubator

• Before Purification Phase 1 (page 8) o Rehydrate lyszymeo Set up centrifuge

Prep. For SDS Gel labPrep. For SDS Gel labcheck for supplies95 water bath

pGLO Bacterial pGLO Bacterial TransformationTransformation

Introduction to TransformationTransformation Laboratory

Data Collection and AnalysisCalculate Transformation Efficiency

Background Background informationinformation

• Genetic transformation:Occurs when a cell takes up and expresses a new piece of genetic material-DNA

• pGLO plasmid contains GFP gene and the gene for beta-lactamase, the ampicillin resistant gene

• Transformed cells are found on the LB/amp and LB/amp/ara plates

Lab OverviewLab Overview

Transformation TipsTransformation Tips• Transform cells and spread plates

o Be careful when handling E. coli: It could cause food poisoning- Decontaminate work surfaces every day and after any spill- All contaminated liquid or solid wastes are decontaminated before

disposal- All persons must wash their hands after handling bacteria and before

exiting the labo DO NOT mouth pipet, eat, or drink while in labo Wear protective eyewear and gloveso Use a 10% bleach solution for at least 20 min for sterilization of loops and

pipets

• Observe transformants and controls/Analyze and interpret resultso Observe the color and glow of the bacteria under a UV lighto Fewer colonies of bacteria if amp negatively affected growtho Equal numbers of colonies on both LB nutrient agar and LB/amp agar if amp

had no effect o The presence of any colonies on the amp plate would suggest that those

bacteria are resistant to the antibiotic ampicillin

Extension ActivityExtension Activity

Green Fluorescent Green Fluorescent Protein PurificationProtein Purification

Genetic Transformation ReviewInoculation- Growing a Cell Culture

Purification Phase 1Purification Phase 2Purification Phase 3

Background Background InformationInformation

• pGLO plasmid contains GFP gene and the gene for beta-lactamase, the ampicillin resistant gene

• When bacteria was plated onto LB agar containing arabinose (LB/amp/ara), GFP was expressed

• When bacteria was plated onto LB agar that did not contain arabinose (LB/amp), the gene was turned off

• Binding Wash Elution

Chromatography Chromatography OverviewOverview

Purification TipsPurification Tips• Purification Phase 1Bacterial Concentration and

Lysiso Centrifugation is a technique used to separate molecules on the basis

of size by high speed spinningo Centrifugation results in a pellet of bacteria found at the bottom of the

tubeo The liqu

• Purification Phase Removing Bacterial Debriso This final centrifugation step serves to separate the large particles of

lysed bacteria (such as the cell membrane and walls) from the much smaller proteins, including GFP

o The supernatant will fluoresce bright green upon exposure to UV lighto Carefully remove the supernatanto id that resides above the pellet is called the supernatant

Purification TipsPurification Tips• Purification Phase 3 Protein Chromatography

o Bacteria contain thousands of endogenous proteins from which GFP must be separated

o In chromatography, a cylinder, or column is densely filled with a “bed” of microscopic beads that will form a matrix through which proteins must pass before being collected

o GFP “sticks” to the column, allowing it to be separated from the bacterial contaminants

o Equilibration buffer Binding buffer Wash buffer Elution buffer

GFP Protein on GFP Protein on Polyacrylamide Polyacrylamide

Gels Gels

Polyacrylamide gelsPolyacrylamide gels• Tutorial 23 and 25 from disc.

Background Background Information/TipsInformation/Tips

• Pages 2-8• Only 4 stations (1, 3, 5, 7)