Download - AP Bio: Wednesday, 2.16.11 Planning Lab 6A; Mutations

Homework: Study for Test on Molecular Genetics

Do Now: Get a copy of Lab 6A: Transformation

Today’s Goals: Design a procedure for genetically engineering E. coli cells to glow. Describe the effects of different types of mutations.

Agenda: Mini-Lecture: Intro to Lab 6A – Transformation Explanation of Lab Report In Lab Groups:

(1) Use the colored cards to make a flow chart and then a procedural list for completing this lab.

(2) Complete the Computer-Based Mutations Lab. Print and hand to me when finished.

Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard College.

AP Biology Lab 6:Genetic Engineering

viaBacterial Transformation

Making E. coli glow like jellyfish

Amy Dickson, Prospect Hill Academy Charter School

All images by Christine Rodriguez and Amy Dickson


GOALS OF THIS LAB PROJECT:

• Make E. coli bacteria glow like jellyfish

• By inserting the GFP (green fluorescent protein) gene from a jellyfish into a bacterial plasmid

• Control when the bacteria express this protein

• By connecting the GFP gene to an “on/off” switch that causes it to be expressed only in certain environments


WHY SHOULD WE DO THIS?

Genetic Engineering is now widely used:

• Bacteria that produce human insulin

• Corn that produces insecticide

• Rice that produces extra vitamin A

• Goats that produce spider silk



To SEE the Central Dogma in action:

DNA RNA Protein

Trait

GFP Gene

• found in jellyfish

• engineered into bacteria

Green Fluorescent Protein

GLOWING CELLS



To SEE the Central Dogma in action:

DNA RNA Protein

Trait

To understand how gene expression is regulated - how cells (and the scientists who manipulate them) control when genes are turned on/off.

onX Trait


A BIT OF BACKGROUND ON GENE REGULATION

Promoter:• A short DNA sequence upstream of a gene where RNA pol. binds to start transcription• Serves as the on/off switch for the gene blocking it turns the gene offWhy do this?•Making proteins only when needed saves energy and materials


A BIT OF BACKGROUND ON GENE REGULATION

promotergenes for arabinose-digesting enzymes

genes not expressed

Ara repressor (active) Arabinose sugar

binding site

Arabinose sugar (inducer)

genes expressed!

Example:• Arabinose is a sugar that bacteria can digest•But no need to make enzymes unless arabinose is around•Normal condition: Promoter blocked by Ara repressor•In presence of arabinose: repressor is inactivated; gene is turned on


QUICK REVIEW

Promoter -

Plasmid -

Transformation - a process in which bacteria take up DNA from their environment

a small, circular piece of bacterial DNA that is not part of the chromosome

an “on/off” switch for a gene

- can be triggered by electric shock or heat shock


STARTING MATERIALS

Bacterial chromosome

E. coli cells

• sensitive to antibiotics

• can’t glow

• competent - able to be transformed


STARTING MATERIALS

AmpR

Ara

promoter

Plasmid containing:

• Ampicillin resistance gene (always expressed)

• Ara promoter - turned on in the presence of

arabinose


STARTING MATERIALS

GFP gene

Jellyfish DNA

GFP = Green Fluorescent Protein

glows under UV light


GFP

Jellyfish DNA

STARTING MATERIALS

E. coli cells

AmpR

Ara

Plasmid


END RESULT

AmpR

Ara

GFP

Recombinant Bacteria…

… that can GLOW!

GROW ON AN AGAR PLATE


HOWEVER…things are actually a bit more

complex.

AmpR

Arapromoter

GFP

pGLO plasmid

makes all transformed bacteria resistant to ampicillin

controls GFP gene expression

only turned on in the presence of arabinose


YOUR TASK:Design an experimental procedure for genetically engineering glowing

bacteria.

pGLO

Goals to consider:

#1 - Make recombinant bacteria

#2 - Select for only the recombinant bacteria

#4 - Establish a control for your experiment to demonstrate that it’s the plasmid that causes ampicillin resistance and the ability to glow.

#3 - Make the recombinant bacteria glow only when we want them to.

Download - AP Bio: Wednesday, 2.16.11 Planning Lab 6A; Mutations

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