Learning TargetsLearning Targets“I Can . . .”“I Can . . .”

-Explain what it means for a gene to be -Explain what it means for a gene to be expressed.expressed.

-Explain the role of plasmids.-Explain the role of plasmids.

-Define bacteria “transformation.”-Define bacteria “transformation.”

-Insert a plasmid into bacteria to -Insert a plasmid into bacteria to observe the process of transformation observe the process of transformation as an important biotechnology process.as an important biotechnology process.

-Define bacteria “transformation.”-Define bacteria “transformation.”

Bacterial Bacterial Transformation Transformation Using the pGLO Using the pGLO

genegeneA Study of Gene ExpressionA Study of Gene Expression

Biology and Honors Biology and Honors Biology:Biology:

Start reading through the Start reading through the experiment procedures experiment procedures on your tables so that you on your tables so that you are familiar with what we are familiar with what we will be doing today.will be doing today.

Background and ReviewBackground and Review

A gene is a piece of DNA that A gene is a piece of DNA that contains the instructions for making contains the instructions for making a particular protein; this protein a particular protein; this protein gives an organism a certain traitgives an organism a certain trait

Some genes can be inserted into Some genes can be inserted into organisms to change their traits; this organisms to change their traits; this is called . . .is called . . .

TRANSFORMATION

Anatomy of a Bacteria Anatomy of a Bacteria CellCell

Bacteria contain bits of circular DNA Bacteria contain bits of circular DNA called plasmidscalled plasmids

Bacteria can exchange plasmids Bacteria can exchange plasmids freely with one another and with freely with one another and with other sourcesother sources

Background and ReviewBackground and Review

You will be working with a bacteria called You will be working with a bacteria called E. E. colicoli

In order to transform In order to transform E. coliE. coli, you need to grow , you need to grow LOTS of itLOTS of it

Today we will be placing Today we will be placing E. coliE. coli on a petri dish on a petri dish and incubating it at body temperature (37◦C) and incubating it at body temperature (37◦C) to grow colonies. to grow colonies.

Each 1mm colony consists of millions of Each 1mm colony consists of millions of bacteria cells originating from a single cell bacteria cells originating from a single cell that has divided through mitosis for several that has divided through mitosis for several generationsgenerations

What is a colony?What is a colony?

How do we get colonies?How do we get colonies?By Preparing A Streak By Preparing A Streak

PlatePlate

Purpose of a Streak PlatePurpose of a Streak Plate

What is aseptic What is aseptic technique?technique?

Be as sterile as possible!Be as sterile as possible!

*Wipe the surface you are working on *Wipe the surface you are working on beforebefore and and afterafter the lab the lab

*Sterilize your instruments with *Sterilize your instruments with extreme heat (or use disposable extreme heat (or use disposable instruments)instruments)

*Open the petri dish lid slightly; do *Open the petri dish lid slightly; do NOT lay the lid on the tableNOT lay the lid on the table

What is aseptic What is aseptic technique?technique?

Why aseptic technique?Why aseptic technique?

What will you do in lab?What will you do in lab? Green Fluorescent Protein (GFP) causes the Green Fluorescent Protein (GFP) causes the

jellyfish species jellyfish species Aequorea victoriaAequorea victoria to glow in the to glow in the dark in the presence of UV light.dark in the presence of UV light.

This gene will be inserted into bacteria, in the This gene will be inserted into bacteria, in the form of a plasmid, to cause them to glow as well form of a plasmid, to cause them to glow as well

What is a plasmid?What is a plasmid?

A A plasmidplasmid is … is … a small circular DNA a small circular DNA

strand in the strand in the cytoplasm of a cytoplasm of a bacterium that can be bacterium that can be moved around from moved around from one cell to another. one cell to another.

Plasmids allow bacteria Plasmids allow bacteria transformation transformation to to occur.occur.

What is a plasmid?What is a plasmid?

What will you do in lab?What will you do in lab?

The pGLO plasmid contains two genes:The pGLO plasmid contains two genes:1.1. The gene for GFP (glowing gene)The gene for GFP (glowing gene)

2.2. The gene to be resistant to ampicillin (an The gene to be resistant to ampicillin (an antibiotic)antibiotic)

Two pGLO tubes in lab: Two pGLO tubes in lab:

-pGLO= no pGLO plasmid-pGLO= no pGLO plasmid

+pGLO = pGLO plasmid+pGLO = pGLO plasmid

For the GFP to actually glow, the sugar arabinose For the GFP to actually glow, the sugar arabinose must be present. must be present.

LB Petri Dish (-pGLO)LB Petri Dish (-pGLO)

LBLB is a nutrient that allows is a nutrient that allows E. coli E. coli to to growgrow

LB/amp Petri Dish (-LB/amp Petri Dish (-pGLO)pGLO)

LBLB is a nutrient that allows is a nutrient that allows E. coli E. coli to to growgrow

Amp stands for Amp stands for ampicillinampicillin, an , an antibioticantibiotic

What do antibiotics do to bacteria What do antibiotics do to bacteria growth?growth?

LB/amp Petri Dish LB/amp Petri Dish (+pGLO)(+pGLO)

LBLB is a nutrient that allows is a nutrient that allows E. coli E. coli to to growgrow

Contains Contains ampicillinampicillin Contains Contains plasmidplasmid with two genes with two genes

LB/amp/ara Petri Dish LB/amp/ara Petri Dish (+pGLO)(+pGLO)

LBLB is a nutrient that allows is a nutrient that allows E. coli E. coli to to growgrow

Contains Contains ampicillinampicillin, an antibiotic, an antibiotic Also contains Also contains arabinosearabinose, a sugar, a sugar Contains Contains plasmidplasmid with two genes with two genes

Review QuestionsReview Questions

1.1. On which of the plates would you On which of the plates would you expect to find bacteria most like the expect to find bacteria most like the original non-transformed original non-transformed E. coliE. coli on on your starter plates? Explain your your starter plates? Explain your predictions. predictions.

LB –pGLOLB –pGLO

LB/amp –pGLOLB/amp –pGLO

LB/amp +pGLOLB/amp +pGLO

LB/amp/ara +pGLOLB/amp/ara +pGLO

Review QuestionsReview Questions

2. If there are any genetically 2. If there are any genetically transformed bacterial cells, on which transformed bacterial cells, on which plate(s) would they most likely be plate(s) would they most likely be located? Explain your predictions.located? Explain your predictions.

LB –pGLOLB –pGLO

LB/amp –pGLOLB/amp –pGLO

LB/amp +pGLOLB/amp +pGLO

LB/amp/ara +pGLOLB/amp/ara +pGLO

Review QuestionsReview Questions

3.3. Which plates should be compared Which plates should be compared to determine if any genetic to determine if any genetic transformation has occurred? transformation has occurred? Why?Why?

LB –pGLOLB –pGLO

LB/amp –pGLOLB/amp –pGLO

LB/amp +pGLOLB/amp +pGLO

LB/amp/ara +pGLOLB/amp/ara +pGLO

Review QuestionsReview Questions

4. What is meant by a control plate? 4. What is meant by a control plate? What purpose does a control serve?What purpose does a control serve?

Hypothesis:Hypothesis:

ReferencesReferences https://sarahinbiology.wordpress.com/2012/05/04/pglo-tran

sformation/ http://www.bio-rad.com/en-us/sku/166-0405edu-pglo-plasm

id https://

stonybrook.digication.com/mariam_shareef/Effects_of_Temperature_on_Bacteria/published

http://lem.ch.unito.it/didattica/infochimica/2008_GFP/lem.ch.unito.it/didattica/infochimica/2008_GFP/immagini/Jellyfish.jpgimmagini/Jellyfish.jpg

https://www.pinterest.com/pin/153826143494514820/https://www.pinterest.com/pin/153826143494514820/ http://dentlight.com/faqs/doe-work/http://dentlight.com/faqs/doe-work/ http://proteopedia.org/wiki/index.php/Image:GFP_mice.pnghttp://proteopedia.org/wiki/index.php/Image:GFP_mice.png