ap biology - r. wingerden · bacterial transformation 6-i in compbook ... ... bacterial...

Investigation 8: Biotechnology Bacterial Transformation

Pearson Education, Inc., publishing as Person Benjamin Cummings College Board, AP Biology Curriculum Framework 2012-2013

Copyright © Rebecca Rehder Wingerden

AP Biology Investigation 8: Bacterial TransformationLearning Objectives • To demonstrate the universality of DNA and its

expression. • To explore the concept of phenotype expression in

organisms. • To explore how genetic information can be transferred

from one organisms to another. • To investigate how horizontal gene transfer is a

mechanisms by which genetic variation is increased in organisms.

• To explore the relationship between environmental factors and gene expression.

• To investigate the connection between the regulation gene expression and observed differences between individuals in a population or organisms.

Copyright © 2012 Rebecca Rehder Wingerden

Complete the following before conducting this investigation:

I. Read Investigation 8: Biotechnology: Bacterial Transformation

II. Answer the following PreLab questions in your Comp Book:

1. Summarize what you will be doing in this investigation.

2. What is the primary question you will be trying to answer in this investigation?


PreLab

Investigation 8: Bacterial Transformation

Getting Started: • Complete LabBench Activity Lab 6 Molecular Biology

Bacterial Transformation 6-I in CompBook (handout) - http://www.phschool.com/science/biology_place/

labbench/lab6/intro.html • Complete Activity - Cloning Paper Plasmid with your

group then each group member should answer questions 1-9 in your Comp Book (handout)

BozemanScience.com: AP Biology Lab 6 - Molecular Biology (9:00 min.) http://www.bozemanscience.com/ap-bio-lab-6-molecular-biology


PreLab


BioRad pGLO Transformation: •Lesson 1 Introduction to Transformation (PreLab) - Read Introduction to Transformation p. 32 •Lesson 1 Focus Questions (PreLab) - Complete Consideration 1: Can I Genetically

Transform an Organism? Which Organism? 4Q p. 33

- Read Consideration 3: The Genes p. 35 - Read Consideration 4: The Act of Transformation

p. 35



Background • DNA provided the instruction necessary of the survival,

growth, and reproduction of an organism. • When genetic information changes, either through

natural processes or genetic engineering, the results may be observable in the organisms.

• Bacterial transformation is of central importance in molecular biology for it allows the introduction of genetically engineered or naturally occurring plasmids in bacterial cells.



Background • Restriction enzymes

cut DNA at specific nucleotide sequences

• DNA fragments cut with a specific restriction enzyme can be spliced into a plasmid that has been cut by the same restriction enzyme forming recombinant plasmid.



Background ~ The Plasmid • In this investigation, you will use a plasmid called

pGLO to transform E. coli. • The transformed bacteria will acquire antibiotic

resistance and glow green when exposed to UV light.

The pGLO Plasmid contains the GFP gene


Transformed E. coli colonies with pGLO

What is a plasmid? A circular piece of autonomously replication DNA. May express a gene for antibiotic resistance or be modified to express proteins of interest.


Background ~ The Gene • GFP is used as a tracer molecule.

Douglas Prasher was the first person to realize the potential of GFP as a tracer molecule. In 1987, he got the idea that sparked the GFP revolution. He thought that GFP from a jellyfish could be used to report when a protein was being made in a cell. Proteins are extremely small and cannot be seen, even under an electron microscope. However, if one could somehow link GFP to a specific protein, for example hemoglobin, one would be able to see the green fluorescence of the GFP that is attached to the hemoglobin. It would be a bit like attaching a light bulb to the hemoglobin molecule. Doug Prasher envisioned that it would be possible to use biomolecular techniques to insert the GFP gene at the end of the hemoglobin gene, right before the stop codon. When the cell needed to make hemoglobin, it would go to the hemoglobin gene, use the information encoded in the gene to make it, but instead of stopping when the hemoglobin was made, this cell would carry on making GFP until it reached the stop codon at the end of the GFP gene. As a result, the cell would produce a hemoglobin molecule with a GFP attached to it




Green Fluorescent Protein (GFP) and GFP-like proteins have become the microscope of the twenty-first century. GFP can show us when a protein is made, and what its movements are (Green Fluorescent Protein: A Molecular Microscope).


Background ~ Gene Expression: • Beta Lactamase - Ampicillin resistance • Green Fluorescent Protein (GFP) - Aequoria victoria

jellyfish gene • araC regulator protein - regulates GFP transcription


Investigation 8: Bacterial TransformationBackground ~ Transcriptional Regulation


RNA Polymerase

Z Y A

Z Y ALacI

Effector (Lactose)

Z Y ALacI

lac Operon

B A DaraC

B A DaraC

RNA Polymerase

Effector (Arabinose)

araC B A D

ara Operon

RNA Polymerase

araC

ara GFP Operon

GFP Gene

araC GFP Gene

araC GFP Gene

Effector (Arabinose)

Operon: The Basic Concept. Cells control metabolism by regulating enzyme activity of the expression of genes coding for enzymes. In bacteria, genes are often clustered into operons, with one promoter serving several adjacent genes. An operator site on the DNA switches the operon ON or OFF, resulting in coordinated regulation the genes.

Inducible operon because they are usually OFF but can be stimulated (induced) when Effector is present. Binding of a specific Effector turns ON transcription. (Negative Gene Regulation- gene is typically OFF).



Investigation 8: Bacterial TransformationWhat is Transformation: Uptake of foreign DNA, often a circular plasmid.

Beta lactamase (ampicillin resistance)

pGLO plasmids

Bacterial chromosomal DNA

Cell wall

GFP

Why Perform Each Transformation Step?

2. Incubate on ice slows fluid cell

membrane !3. Heat-shock Increases permeability

of membranes !4. Nutrient broth

incubation Allows beta-lactamase expression

1. Transformation solution = CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates


Investigation 8: Bacterial TransformationTransformation Procedure Overview: Predicting Results

Day 3

Day 2

Day 1 Day 2

Predict Results- Growth?

LB/amp LB/amp/ arabinose LB/amp LB



Day 3

Day 2

Day 1 Day 2

Transformation Procedure Overview: Predicting Results BioRad pGLO Transformation: •Lesson 1 Focus Questions (Day 1) - Complete Consideration 2: How Can I Tell if Cells Have

Been Genetically Transformed? Observations a-f and 2Q p. 34

•Lesson 2 Transformation Laboratory (Day 1) - Read Transformation Procedure 1-12 p. 37-41 •Lesson 2 Review Questions (Day 1) - Complete 4Q on p. 42 •Lesson 2 Transformation Laboratory (Day 2) - Complete Transformation Procedure 1-12 p. 37-41 - Copy Data Table 1: Transformation of E. coli with pGLO




Table 1: Transformation of E. coli with pGLO

Carefully observe and draw what you see on each of the four plates.

Write down the following observation for each plate:

1. How much bacterial growth do yo used on each plate, relatively speaking? 2. What color are the bacteria? 3. How many bacterial colonies are on each plate (count the spots you see).

BioRad pGLO Transformation: •Lesson 3 Data Collection and Analysis (Day 3) - Complete Data Collection: 4Q, Transformation Plate

Observations, Control Plates Observations p. 43 - Complete Analysis of Results: 4Q p. 44 •Lesson 3 Review Questions (Day 3) - Complete What’s Glowing: 4Q p. 45 - Complete Interaction between Genes

and Environment: 3Q p. 46



ap biology - r. wingerden · bacterial transformation 6-i in compbook ... ... bacterial...

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