Activity 3.1.6: Genetic Engineering: Making EColi ... - GabrielleMilton

Project Lead The Way, Inc.

Copyright 2010

PLTW
TM

-

BE
–

Unit 3
–

Activity 3.1.6
–

Genet
ic Engineering: Making E.Coli Glow Like Jellyfish
–

Page
1



Activity 3.1.6
–

Genetic Engineering:
Making
E. Coli

Glow Like Jellyfish



Introduction

In this activity
you

will perform genetic engineering
–

learning how to tinker w
ith the
nuts and bolts of life.

Initially, working in teams
,

you
will design a sta
rt
-
up biopharma
company.

After creating a name for the company, the start
-
up
company
will be in
r
esearch and development mode.
The methodology to be followed will parallel the
actual development and production stages for insulin production.


The gene to
be inserted for drug production is green fluorescent protein (GFP) from
the jellyfish
Aequorea victoria.

GFP causes the jellyfish to fluoresce green when as a
result

of their own bioluminescence.
They are truly the enlightened ones.


Common approaches to

genetic modification of bacterial include manipulation of cell
membranes w
ith chemicals or electricity.
Bacteria must be in a particular
physiological state before they can be transformed. This state is commo
nly referred
to as
competency
.
This can be achi
eved by th
e addition of a salt solution.
Electroporation of cell membranes is used as a tool in injecting bot
h drugs and DNA
into the cell.
These methods will be investigated by each biopharma company to find
the o
ptimal method for each company’s

bioreacto
rs.


Equipment



Computer



Lab book or journal



Incubator or shaker incubator
(water or air)



Microcentifuge tube racks



Microcentifuge tubes



Flasks



Black lights



Micropipettes



Pipette tips



Colony counter



Hand held counter (clicker)



LB agar and broth media



Petri dishes



CaCl
2



pGlo plasmid



Distilled water


Procedure

In order to demonstrate successful gen
etic engineering the bacterial
drug factories

must express their newly acquired jellyfish gene and produce the fluorescent protein,
Project Lead The Way, Inc.

Copyright 2010

PLTW
TM

-

BE
–

Unit 3
–

Activity 3.1.6
–

Genet
ic Engineering: Making E.Coli Glow Like Jellyfish
–

Page
2

which will cause them to
glow a brilliant green under ultraviolet light. The follow
-
up
components of this

activity include scale
-
up and p
roduction. After a review of
methods of genetic transformation your group will select to either use CaCl
2

and
heat shock, or electroporation (if

available). Continue to follow the daily instructions.


Timeline


Day 1
–

mrepare growth media
*

Date ___________________

Day 2
–

ptart culture of
E. coli

Date___________________

Day 3
–

Transform
E. coli

with pGlo

Date ___________________

Day 4
–

Analy
ze results!

䑡ae __________________


* Media may have already been prepared by instructor


Day 1
–

Preparation of media


Mix and autoclave media for three different conditions to be used:



LB agar



LB agar with ampicillin



LB agar with ampicillin and arabin
ose.


Day 2
-

Inoculation of E. coli


Inoculate starter plates of
E. coli
.
Rehydrate lyophilized culture of
E. coli

(HB 101)
wi
th sterile water or LB broth.
Each biopharma company will start one plate from the
rehydrated culture of
E. coli
.
Streak for isol
ation with a str
erile loop onto the LB
plates.
Incubate at 32 o C for 24
-
36 hours.


Day 3 Transformation:


Method with CaCl
2

and Heat Shock

Transfer of
E. coli




Place 2 sterile micro test tubes in a micr
o tube rack and label one tube
+DNA

and another
-
DNA
.

Both tubes should be labeled with your
biopharma name. Place them in a floating test
-
tube rack.



Using a p
-
1000 and a sterile tip transfer 250 µl of CaCl
2

into each tube and
place tubes in floating test tube rack on ice.



Find two isolated colonies on E. c
oli starter plate of the same size. With
safety glasses on, pick up

one of the colonies of bacteria

from your
starter plate and place the loop into the
+DNA

tube and twist the loop back
and forth between your fingers in the CaCl
2

solution. Place in the flo
ating
rack on ice. Place the used loop in the biohazard waste.



With a new sterile loop, repeat for the

-
DNA

tube.



Rehydrate freeze
-
dried pGlo with 250 µl sterile water or CaCl
2

solution.



Observations

Project Lead The Way, Inc.

Copyright 2010

PLTW
TM

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BE
–

Unit 3
–

Activity 3.1.6
–

Genet
ic Engineering: Making E.Coli Glow Like Jellyfish
–

Page
3



Before doing anything else observe your
E. coli

and
pGLO DNA solution
with the UV
lamp. Note your observations.


DNA Insertion




Immerse a new sterile loop into the plasmid DNA stock tube. Withdraw a
loopful (10 µ). There should be a film of pGlo across the ring. This is should
look like the sheen across a
ring toy used for blowing soap bubbles.




Mix the loopful into the cell suspension of the

+DNA

tube. Close the tube
and return it to the rack on ice. Also close the

-
DNA

tube (NO pGlo.)



Place the tubes on ice for 10 minutes. Make sure the tubes are in con
tact
with the ice bath.



Prepare plates during the incubation. Collect four agar plates with the
following media combinations and label accordingly:

o

LB plate
-

DNA

o

LB/amp plate
-

DNA

o

LB/amp plate
+ DNA

o

LB/amp/ara plate
+ DNA



Heat Shock




Transfer both th
e floating rack of the (+) and (
-
) tubes into the water bath,
set at 42
-
50 °C, for exactly 50 seconds. Biopharma companies can vary
temperature to see which has the highest transformation efficiency.



After 50 seconds, place both tubes back on ice. The
ra
pid

change from the
ice (0 °C) to 50 (?) °C and then back to the ice yields the best
transformation results. Incubate tubes on ice for 2 minutes.


Electroporation




Place 2 sterile micro test tubes in a micr
o tube rack and label one tube
+DNA

and another
-
DNA
.

Both tubes should be labeled with your
biopharma name. Place them in a floating test
-
tube rack.




Using a p
-
1000 and a st
erile tip transfer 250 µl of
ice
-
cold distilled H
2
O into
each tube and place tubes in floating test tube rack on ice.




Find two iso
lated colonies on E. coli s
tarter plate of the same size.
With
safety glasses on, pick up

one of the colonies of bacteria

from your
starter plate and place the loop into the
+DNA

tube and twist the loop back
and forth between your fingers in the cold H
2
O s
olution.
Plac
e in the floating
rack on ice.
Place the use
d loop in the biohazard waste.
With a new sterile
loop, repeat for the

-
DNA

tube.



Rehydrate freeze
-
dried pGlo with 250 µl sterile water or CaCl
2

solution.



Chill cuvette and cuvette holder.

Project Lead The Way, Inc.

Copyright 2010

PLTW
TM

-

BE
–

Unit 3
–

Activity 3.1.6
–

Genet
ic Engineering: Making E.Coli Glow Like Jellyfish
–

Page
4



Just bef
ore electroporation, add a loop of pGlo plasmid (10 µl) to the cells
and mix well. Take an electroporation cuvette. Add the cell and plasmid
preparation to it. (MUST BE DONE RAPIDLY)



Place the cuvette in the electroporator. Set the voltage to 1900 V. Refe
r to
electroporation manual for pulse delivery.


Plating transformants (from heat shock or electroporation)




Take the floa
ting rack out of the ice bath.
Using a sterile pipette transfer
add 250 µl of LB broth to each tube. Incubate both tubes for 10 minu
tes at
room temperature.



Mix the tubes contents by flicking the bottom of the tube with your finger.
Using a new sterile pipette for each tube, pipette 100 µl of the
transformation and control incubations onto the appropriate plates.



Spread the suspensio
ns evenly around the surface of the agar by quickly
streaking the flat surface of a new sterile loop back and
forth across the
plate surface.

Use a new sterile loop for each plate
.



Stack up your plates and tape them together. Write your group name and
clas
s period on the bottom of the stack and
place it upside down

in the 32
°C incubator until the next day.


Transformation efficiency


There are several ways to calculate transformation efficienc
y. What is the best
approach?
Look over all of your plates and
also determine the amount of plasmid
DNA added to do this calculation.


An
oral presentation

will be given covering the experimental design, sketches
,

and
application of

genetic modification.
Your

team will
need to
document results
throughout with digital
photos or graphic animations which will be incorporated into a
PowerPoint presentation.


Conclusion


1.

What are the ethical considerations involved in genetic engineering?

Many people believe that it goes against nature and other beliefs

-
do animals have r
ights? Should they be used in this kind of research?

-
should we eat GMO’s

-
How great are the potential risks?

-
How far is too far? (How far should commercial secrecy be allowed?)

-

Should anyone be able to patent a genetically modified animal or plant?

-

I
s
genetic engineering to make a staple crop more resist in marginal conditions
a potential boon for Third World agriculture, or another danger of increased
dependence on rich "developed" countries?




Project Lead The Way, Inc.

Copyright 2010

PLTW
TM

-

BE
–

Unit 3
–

Activity 3.1.6
–

Genet
ic Engineering: Making E.Coli Glow Like Jellyfish
–

Page
5


2.

What are some beneficial applications of recombinant D
NA technology?

-
Human insulin

-

It can be used in plants to increase their yield to produce nutritional content.

-

Improvements in cancer research

-
produce hormones for females with fertility issues

-
Vaccines

3.

Why is
E. coli

HB 101 a good candidate for a bi
oreactor?

-
It is a resistant strand that keeps a lot of undesired bacteria away but will work well with
the
ampicillan
.



4.

What genes are found on the pGlo plasmid and what are there functions?

-
Green Fluorescent Protein
: glowing gene

-
AmpR
: gene to be resi
stant to ampicillin





5.

How does a genetic engineer
distinguish bacteria containing the plasmid DNA
from those bacteria lacking the plasmid DNA?



-
w
h
ether it grows and glows because those are the two charateristics in the plasmid that
it is supposed to

gi
ve to the bacteria.


6.

How does a genetic engineer determine if the bacterium has truly been
transformed?

-

The bacterium will pick up the trait that the gene gave it when it transformed.

-

the codes have been changed and connected



7.

How does heat shock ass
ist in gene insertion?


Heats shock confuses the membrane

-

it helps by separating the bacteria allowing for the ampicillin and the GFP to be
inserted into the bacteria while

separated.




8.

How does electroporation assist in gene insertion?

-
quick voltage s
hock may disrupt areas of the membrane temporarily, allowing
polar molecules to pass, but then the membrane may reseal quickly and leave
the cell intact.

Project Lead The Way, Inc.

Copyright 2010

PLTW
TM

-

BE
–

Unit 3
–

Activity 3.1.6
–

Genet
ic Engineering: Making E.Coli Glow Like Jellyfish
–

Page
6