Transformation of E.coli with pGLO

Transformation of E.coli with pGLO

Dan Prochaska and Jeff Winslow

Talawanda/Butler Tech

Dan Prochaska and Jeff Winslow

Talawanda/Butler Tech

PurposePurpose

Challenge students to participate in cutting edge laboratory activities that emphasize important skills carried out in a structured environment.

To make connections to modern commercially viable protocols, i.e. human insulin production.

Challenge students to participate in cutting edge laboratory activities that emphasize important skills carried out in a structured environment.

To make connections to modern commercially viable protocols, i.e. human insulin production.

Lesson PlanLesson Plan

Three day lesson. Day 1: Powerpoint introduction, handouts with

protocol, streaking starter plates, and pre-assessment

Day 2: Complete transformation protocol, student groups make predictions about results

Day 3: Observe plates using U.V. pen lights and record observations. Make connections to real world applications. Complete post assessments.

Three day lesson. Day 1: Powerpoint introduction, handouts with

protocol, streaking starter plates, and pre-assessment

Day 2: Complete transformation protocol, student groups make predictions about results

Day 3: Observe plates using U.V. pen lights and record observations. Make connections to real world applications. Complete post assessments.

pGLO lab. ObjectivespGLO lab. Objectives

Transform E. coli host cells with plasmid DNA.

Predict the results on selective agar plates. Observe and record actual plate data. Draw conclusions from the experiment and

list possible sources of error.

Transform E. coli host cells with plasmid DNA.

Predict the results on selective agar plates. Observe and record actual plate data. Draw conclusions from the experiment and

list possible sources of error.

IntroductionIntroduction

What is genetic transformation? When host bacteria acquire novel traits that are

encoded in genes contained on plasmid, (pGLO) DNA.

What new traits will E.coli acquire from the transformation?

Antibiotic resistance (ampicillin)Green Fluorescence: Where did this trait come from?

What is genetic transformation? When host bacteria acquire novel traits that are

encoded in genes contained on plasmid, (pGLO) DNA.

What new traits will E.coli acquire from the transformation?

Antibiotic resistance (ampicillin)Green Fluorescence: Where did this trait come from?

What is the source of green flourescence?

What is the source of green flourescence?

The marine jellyfish Aequorea victoria The marine jellyfish Aequorea victoria

Plasmid DNAPlasmid DNA

Very small (103bp ) piece of circular DNA Replicates autonomously, (ori site). Is in addition to the large (106bp) E. coli

chromosome. Can be exchanged between cells. Carries important genes for traits such as

antibiotic resistance. In this laboratory we are investigating ampicillin

resistance.

Very small (103bp ) piece of circular DNA Replicates autonomously, (ori site). Is in addition to the large (106bp) E. coli

chromosome. Can be exchanged between cells. Carries important genes for traits such as

antibiotic resistance. In this laboratory we are investigating ampicillin

resistance.

Plasmid DNAPlasmid DNA

A small circular piece of DNA that is naturally occurring in some bacteria. It can be exchanged between cells and often carries antibiotic resistance genes

pGLO an engineered plasmidpGLO an engineered plasmid

pGLO plasmid genespGLO plasmid genes

GFP gene encodes a green fluorescent protein (GFP)

Bla gene encodes the enzyme beta lactamase that degrades the antibiotic ampicillin thus, protecting the cell from its harmful effects.

AraC is a regulatory gene that encodes a protein that acts like a switch that turns on the expression of the GFP gene.

GFP gene encodes a green fluorescent protein (GFP)

Bla gene encodes the enzyme beta lactamase that degrades the antibiotic ampicillin thus, protecting the cell from its harmful effects.

AraC is a regulatory gene that encodes a protein that acts like a switch that turns on the expression of the GFP gene.

Transformation- TermsTransformation- Terms

Competence: cells are able to take up a plasmid from their surroundings

How do we make our E.coli cells competent?

Gene expression: cells replicate, transcribe, and translate genes located on the pGLO plasmid

Competence: cells are able to take up a plasmid from their surroundings

How do we make our E.coli cells competent?

Gene expression: cells replicate, transcribe, and translate genes located on the pGLO plasmid

TransformationTransformation

Beta lactamase(ampicillin resistance)

pGLO plasmids

Bacterial chromosomal DNA

Cell wall

GFP