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 with the
nuts and bolts of life. Initially, working in teams, you will design a start-up biopharma
company. After creating a name for the company, the start-up company will be in
research 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 with chemicals or electricity. Bacteria must be in a particular
physiological state before they can be transformed. This state is commonly referred
to as competency. This can be achieved by the addition of a salt solution.
Electroporation of cell membranes is used as a tool in injecting both drugs and DNA
into the cell. These methods will be investigated by each biopharma company to find
the optimal method for each company’s bioreactors.
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
 CaCl2
 pGlo plasmid
 Distilled water
Procedure
In order to demonstrate successful genetic engineering the bacterial drug factories
must express their newly acquired jellyfish gene and produce the fluorescent protein,
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PLTW TM - BE– Unit 3 – Activity 3.1.6 – Genetic Engineering: Making E.Coli Glow Like Jellyfish – Page 1
which will cause them to glow a brilliant green under ultraviolet light. The follow-up
components of this activity include scale-up and production. After a review of
methods of genetic transformation your group will select to either use CaCl2 and
heat shock, or electroporation (if available). Continue to follow the daily instructions.
Timeline
Day 1 – Prepare growth media*
Day 2 – Start culture of E. coli
Day 3 – Transform E. coli with pGlo
Day 4 – Analyze results!
Date ___________________
Date___________________
Date ___________________
Date ___________________
* 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 arabinose.
Day 2 - Inoculation of E. coli
Inoculate starter plates of E. coli. Rehydrate lyophilized culture of E. coli (HB 101)
with sterile water or LB broth. Each biopharma company will start one plate from the
rehydrated culture of E. coli. Streak for isolation with a strerile loop onto the LB
plates. Incubate at 32 o C for 24-36 hours.
Day 3 Transformation:
Method with CaCl2 and Heat Shock
Transfer of E. coli
 Place 2 sterile micro test tubes in a micro 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 CaCl2 into each tube and
place tubes in floating test tube rack on ice.
 Find two isolated colonies on E. coli 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 CaCl2 solution. Place in the floating
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 CaCl2 solution.
Observations
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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 contact
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 the 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 rapid 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 micro 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 ice-cold distilled H2O into
each tube and place tubes in floating test tube rack on ice.
 Find two isolated colonies on E. coli 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 cold H2O solution. Place in the floating
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 CaCl2 solution.
 Chill cuvette and cuvette holder.
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 Just before 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. Refer to
electroporation manual for pulse delivery.
Plating transformants (from heat shock or electroporation)
 Take the floating 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 minutes 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 suspensions 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
class 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 efficiency. 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?
You are altering living things on a genetically level and people could argue god made
them like that and you change them.
2. What are some beneficial applications of recombinant DNA technology?
You can remove bad genes of an organism and replaced them with beneficial genes.
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3. Why is E. coli HB 101 a good candidate for a bioreactor?
This strain of e coli does not make people sick and can grow easily in every
environment
4. What genes are found on the pGlo plasmid and what are there functions?
The genes in the pGlo plasmid use the energy from the cell in order to make it glow.
5. How does a genetic engineer distinguish bacteria containing the plasmid DNA
from those bacteria lacking the plasmid DNA?
The bacteria with the plasmid in theory should be able to glow in the dark using a UV
light.
6. How does a genetic engineer determine if the bacterium has truly been
transformed?
You can see the new DNA codes in the bacteria and see if it experiences the new trait.
7. How does heat shock assist in gene insertion?
The heat shock opens up the pores of the bacteria cells and allows DNA info to enter
the bacteria easier.
8. How does electroporation assist in gene insertion?
It adds a charge that stretches the bacteria too allow DNA to enter it easily and stay in
there.
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PLTW TM - BE– Unit 3 – Activity 3.1.6 – Genetic Engineering: Making E.Coli Glow Like Jellyfish – Page 5