Ap bio wednesday 2 16 11 planning lab 6a mutations
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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.

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

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Ap bio wednesday 2 16 11 planning lab 6a mutations

AP Bio: Wednesday, 2.16.11Planning 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.


Ap biology lab 6 genetic engineering via bacterial transformation

AP Biology Lab 6:Genetic EngineeringviaBacterial Transformation

Making E. coli glow like jellyfish

Amy Dickson, Prospect Hill Academy Charter School

All images by Christine Rodriguez and Amy Dickson


Ap bio wednesday 2 16 11 planning lab 6a mutations

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


Ap bio wednesday 2 16 11 planning lab 6a mutations

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


Ap bio wednesday 2 16 11 planning lab 6a mutations

DNA

RNA

Protein

Trait

Green Fluorescent Protein

  • GFP Gene

  • found in jellyfish

  • engineered into bacteria

GLOWING CELLS

WHY SHOULD WE DO THIS?

To SEE the Central Dogma in action:


Ap bio wednesday 2 16 11 planning lab 6a mutations

DNA

RNA

Protein

Trait

WHY SHOULD WE DO THIS?

To SEE the Central Dogma in action:

X

on

Trait

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


Ap bio wednesday 2 16 11 planning lab 6a mutations

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 off

  • Why do this?

  • Making proteins only when needed saves energy and materials


Ap bio wednesday 2 16 11 planning lab 6a mutations

A BIT OF BACKGROUND ON GENE REGULATION

  • 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

genes for arabinose-digesting enzymes

promoter

genes not expressed

Ara repressor (active)

Arabinose sugar binding site

genes expressed!

Arabinose sugar (inducer)


Ap bio wednesday 2 16 11 planning lab 6a mutations

QUICK REVIEW

Promoter -

Plasmid -

Transformation -

an “on/off” switch for a gene

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

a process in which bacteria take up DNA from their environment

- can be triggered by electric shock or heat shock


Ap bio wednesday 2 16 11 planning lab 6a mutations

STARTING MATERIALS

E. coli cells

  • sensitive to antibiotics

  • can’t glow

  • competent - able to be transformed

Bacterial chromosome


Ap bio wednesday 2 16 11 planning lab 6a mutations

AmpR

Ara

promoter

STARTING MATERIALS

  • Plasmid containing:

  • Ampicillin resistance gene (always expressed)

  • Ara promoter - turned on in the presence of arabinose


Ap bio wednesday 2 16 11 planning lab 6a mutations

GFP gene

STARTING MATERIALS

Jellyfish DNA

GFP = Green Fluorescent Protein

glows under UV light


Ap bio wednesday 2 16 11 planning lab 6a mutations

AmpR

Ara

GFP

STARTING MATERIALS

E. coli cells

Plasmid

Jellyfish DNA


Ap bio wednesday 2 16 11 planning lab 6a mutations

AmpR

GROW ON AN AGAR PLATE

GFP

Ara

… that can GLOW!

END RESULT

Recombinant Bacteria…


Ap bio wednesday 2 16 11 planning lab 6a mutations

makes all transformed bacteria resistant to ampicillin

controls GFP gene expression

only turned on in the presence of arabinose

HOWEVER…

things are actually a bit more complex.

AmpR

pGLO plasmid

GFP

Ara

promoter


Ap bio wednesday 2 16 11 planning lab 6a mutations

pGLO

YOUR TASK:

Design an experimental procedure for genetically engineering glowing bacteria.

Goals to consider:

#1 - Make recombinant bacteria

#2 - Select for only the recombinant bacteria

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

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


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