PGLO Outbreak

Lauren Bertelson, Danielle Honan, Hannah Gorman, Kiera Jost

10/3/2016
Summary
What is PGLO?
PGLO is a plasmid

What is a plasmid?
A circular piece of
independently replicating
DNA
Can express an antibiotic
resistant gene or proteins
of interests

PGLO gene expression:

Beta Lactamase Ampicillin
resistance
GFP jellyfish gene
araC regulator protein
*photo via in-class presentation
regulates GFP
transcription
Green Fluorescent Protein
The pathogen glows
because of Green
Fluorescent Protein, or
GFP, a gene in the pGLO
plasmid

GFP has existed for millions

of years in Aequorea
victoria, a species of
jellyfish

*photo via in-class presentation

GFP
In Aequorea victoria, aequorin (a protein) releases blue light

The GFP absorbs this blue light, which gives off the glow that characterizes GFP

The team that discovered and developed GFP received a Nobel Prize for Chemistry in
2008

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/images/alba2.jp
g
Uses for GFP
GFP serves as a biological marker and allows scientists to
localize and regulate gene expression

There are several types of biological markers:

Gene marker: GFP is paired with a specific gene to show where the
gene is expressed (applies to research)

Cell marker: GFP is paired with a specific type of cells to show their
location and how they divide (also applies to research)

Selectable marker: GFP is included in a plasmid to confirm that a

microbe receives a genetic insert (applies to pharmaceuticals)
https://stickyends.files.wordpress.com/2009/05/gfp1.gif?w=300&h=185
Lab Procedure

*photos via in-class presentation

Our Experiment
Hypotheses: Experimental Design:
Double dash agar causes the
bacteria to glow
Organism D glows because it
is a different species than
A, B, and C

Predictions:
If bacteria A, B, C, and D are
all the same species and
are placed on double dash
agar, they will all glow
If bacteria D is different than
A, B, and C, it only will
glow on double dash agar
If bacteria D glows on single
dash agar, it will glow if
the food is not the reason
Our Results
Observations:

Bacteria C and D both glow on

double dash agar, while A
and B do not

Bacteria C and D both glow

lightly on single dash agar,
but not very bright.

This could be a result of

cross contamination
Why do C and D glow on double dash
agar?
Double dash agar
contains a
monosaccharide
called arabinose

Arabinose is an
effector to the
PGLO genes ara
GFP Operon
This means that when
arabinose is
present, the GFP
gene is activated
through
*photo via in-class presentation
transcriptional
regulation
Antibiotic
Resistance
Antibiotic Resistance
The pGLO plasmid has a section of DNA called Beta Lactamase.

Beta Lactamase causes ampicillin resistance.

Organism A and B don't have the pGLO plasmid, so they do not have
antibiotic resistance.

Organism C and D contain the pGLO plasmid and are able to resist the
effects of the antibiotic.

Single dash agar and double dash agar both contain ampicillin.
Organism A and B are both unable to grow on it.
Chromatography
Chromatography
Chromatography is used in
purifying one protein of interest
from other naturally occurring
e.coli gene product

To purify GFP, we use column

chromatography which is
specially used in purifying
proteins

Three types:
Size Exclusion
*photo via in-class presentation
Ion Exchange
Column Chromatography- Hydrophobic
Interaction
Substances are hydrophobic when
they do not interact with water

The process of column

chromatography separates a single
recombinant protein from E. coli
gene products using hydrophobic
interaction

Hydrophobic interaction
Chromatography (HIC): *photo via in-class presentation

a separation technique that uses

Steps
Step 1: Add bacterial lysate to column matrix in high salt buffer

Hydrophobic proteins react with column and less hydrophobic proteins interact with
salt ions and H20.

Step 2: Using low salt buffer, wash the less hydrophobic proteins from the
column.

The proteins that did not react with the column (less hydrophobic E.coli proteins)
will fall from the column

GFP remains bound to the column because of its hydrophobicity

Step 3: Add a no salt buffer to elute GFP from column.

GFP is released from column matrix and flows through column

*photos via in-class presentation

Disease Info
Dr. Vass
Spread through the stolen thermos
The thermos contained liter of the virus

1 mL can infect 100,000 people

Can only be spread through contact

Not airborne

No other reported cases

Symptoms:
Zombism

Hair growth Dr. Vass

Zombie Schoep
Spread through tainted chicken from Chipotle

Symptoms experienced:
Twitching

Groaning

Progression of symptoms: moved up from feet

Zombie Schoep
Contain the
Situation
How to Stop Contamination
Quarantine each person in a quarantine If you exhibit symptoms do not go out in
hospital room. public, stay in your house and call an
ambulance.
Send out a memo like an amber alert to
notify the public. Try not to leave the house and close
schools until the disease is contained.

http://abcnews.go.com/Technology/blizzard-wireless-emer http://www.dailymail.co.uk/news/article-2788717/new-york-hos
gency-alerts-people/story?id=18434213 pitals-sending-actors-fake-ebola-symptoms-test-response-dead
ly-virus.html
Distribution of Cure
Use affinity chromatography to isolate GFP and create a vaccination based off of the
information gathered.

Distribute cure through a vaccine.

Opposed to distributing vaccine in a center, go house to house and distribute to prevent

further contamination.

https://celiac.org/celiac-disease/understanding-ce
liac-disease-2/celiac-disease-vaccinations/
Bibliography
Bibliography
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/cooluses1.html

http://www.pbs.org/wgbh/nova/bioterror/

http://pdb101.rcsb.org/motm/42

https://en.wikipedia.org/wiki/Column_chromatography