AP Biology
BACTERIAL TRANSFORMATION LAB
Lab Procedures, Rationale, and Protocol
Please read the pdf file with the full lab description that can be found on the class website, in
the AP Biology C Page
http://merrillville.schoolwires.net/Page/5435
The full lab protocol is listed by the title “bacterial transformation lab”
o An overview of the procedure and protocols begins on page 9 of the pdf file
o The step by step procedure begins on page 12 of the pdf file
o We will be using traditional procedures, not the Optional Rapid Transformation
IT IS EXPECTED THAT ALL STUDENTS FULLY READ AND REVIEW THE PROCEDURES AS
DESCRIBED IN THIS PDF FILE. IT IS YOUR RESPONSIBILITY TO UNDERSTAND WHAT YOU WILL BE
EXPECTED TO DO AND WHY EACH STEP IS A NECESSARY PART OF THE PROCEDURE.
A lab handout will be provided for you, but there is also a word file with the lab handout. The
title, not surprisingly, is Transformation Lab Handout
Genetic Engineering Basics
One of the most common applications of genetic engineering is the transformation of bacteria
to create new strains of bacterial cells that will produce some useful protein.
The simple nature of bacteria allows the cells to be cultured in large quantities using minimal
space and resources. Also the nature of bacterial genetics simplifies the process of
transformation and the ability to control the genetic activity of the transformed cells.
◦ Bacteria commonly absorb and exchange plasmids, so the use of plasmids as a vector is fairly simple to
accomplish
◦ Bacterial genes are part of an operon which allows control of the expression of the gene. For example,
associating the recombinant gene with the lac operon would allow you to control the expression of the
gene by controlling the food supply in the culture. Providing the cells with lactose (and denying the
cells a source of glucose) would maximize the activity of the operon and cause the cells to produce the
desired protein in large quantities
◦ Plasmids commonly contain genes for antibiotic resistance, which provides us with a simple method of
isolating the transformed cells and eliminating all cells that were not transformed.
Producing Recombinant Bacteria
1. Choose an appropriate vector
2. Prepare the vector for receiving the donor gene
3. Isolate the donor gene
4. Insert the donor gene into the vector
5. Prepare the host cells to receive the vector
6. Introduce the vector & transform the host cells
7. Isolate the transformed cells
Donor DNA
In our experiment, the donor gene will be a
gene isolated from jellyfish. The gene is for
fluorescence, so the transformed cells will gain
the ability to produce GFP (green fluorescent
protein). The cells will glow green when
exposed to ultraviolet light
Preparing the Vector
Both the plasmid and the donor DNA will be cut with the same restriction enzyme. This will
leave both donor and vector DNA with complementary “sticky ends”
The choice of an appropriate restriction enzyme is vital. A number of factors must be taken into
consideration for success
◦ The plasmid must have only one restriction site for the enzyme you use, more than one and the plasmid
will not be “opened” it will be destroyed
◦ The donor gene must be flanked on both ends by restriction sites so that it will “stick” to the plasmid at
both ends, completing the circular nature of the plasmid
◦ The donor gene must not have any restriction sites within it, or the gene itself will be destroyed
Once the donor gene and the plasmid are cut and combined, they must be permanently
attached by using DNA ligase (remember that DNA ligase serves the natural function of binding
okazaki fragments during replication).
Transformation
Even though bacteria will accept plasmids naturally, success is not guaranteed. A number of
steps must be undertaken to maximize the rate of transformation
First, bacteria are not “competent” to receive plasmids throughout their entire life cycle. Well
established colonies of bacteria will not be transformed. Timing is of the utmost importance.
The colonies will only be competent to receive plasmids if they are in the “logarithmic growth”
phase of their life cycle. In other words, they will only be competent if they are in their phase of
fastest growth and reproduction (which for our purposes is within 24 hours of the introduction
of the bacteria to the culture media)
Also, bacteria will have a higher rate of transformation if the colonies are stressed. We will
introduce stress in 2 ways, chemical and temperature stress.
◦ Chemical stress will be accomplished by adding a quantity of CaCl2 to the cultures, affecting the
permeability of the cell membranes
◦ Temperature stress will be accomplished by heat shocking the cultures – moving them rapidly from and
ice bath and a warm water bath
Isolating the Transformed cells
Remember that plasmids generally contain genes that bacterial cells will use for protection
against other microorganisms. The plasmid that we will use for a vector will contain a gene for
ampicillin resistance.
Ampicillin is a common antibiotic. Most students have been given a prescription for ampicillin
to get rid of an infection at some point in their lives (UNLESS THEY ARE ALLERGIC TO IT, IN
WHICH CASE THEY SHOULD NOT HANDLE ANY OF THE MATERIALS THAT CONTAIN THE
AMPICILLIN – A REACTION IS UNLIKELY UNLESS THE AMPICILLIN IS ACTUALLY INGESTED, BUT
WEAR THE GLOVES AND THE GOGGLES AND LET YOUR PARTNERS PLATE OUT THOSE CULTURES).
Only the bacterial cells that successfully take up a plasmid will have the gene for antibiotic
resistance, so all cells that have not been transformed will be killed by the ampicillin. Any cells
that remain will have been transformed, and will be isolated in the “amp+” cultures
Procedure - Preparing the Culture Plates
Each group will need 5 plates. A source plate
for the stock culture and 4 plates for the actual
experiment.
Control Plates
◦ will not receive the recombinant plasmids
- DNA, - amp
(no ampicillin)
- DNA, + amp
(get ampicillin)
Experimental Plates
◦ will receive the recombinant plasmids
+ DNA, - amp
(no ampicillin)
+ DNA, + amp
(get ampicillin)
The plates need to be poured using melted
nutrient agar. We will use sterile, disposable
plastic petri dishes. They may have been
poured ahead of time and provided for you, or
you may be pouring them yourselves.
https://www.youtube.com/watch?v=PiWwnBbCrNs
Each plate will be labelled on the bottom as
described, to show what treatment it will
receive
The plates that receive the ampicillin will also
be “striped” – marked down the side of the lid
to indicate that they have had ampicillin added
to the agar
Procedure Day 1 - Prepare Source Plates
Sterile technique is absolutely necessary.
Transfer a “bacto-bead” to the petri dish, allow it
to melt, and use the sterile inoculating loop to
streak the sample. Rotate the dish 45 degrees
and streak across the pattern you already made.
The streak pattern should be similar to the
sample shown on the right
The following video gives a decent explanation of
the process, but we will be using disposable,
sterile plastic inoculating loops instead of reusable metal loops, so we will NOT be flame
sterilizing them. After use they go directly into
the bleach bucket. Don’t set them on the table.
https://www.youtube.com/watch?v=Ay2hhujTuvg
Please note that this culture has had time to incubate.
You will not see established colonies when you streak
your plate!
Preparing Cultures for Transformation
Materials:
◦
◦
◦
◦
1.
Source plate with competent E. coli colonies
Ice Bath
Heat Shock bath (42oC)
3 microcentrifuge tubes
Using a sterile inoculating loop, transfer
approximately 8-10 colonies from the source
plate to the “- DNA” microtube with .50 mL
of CaCl2. (colonies only, not the agar!!)
2.
◦ 1 with .50 mL CaCl2 labelled –DNA
◦ 1 empty tube labelled + DNA
◦ 1 with the prepared recombinant plasmids (“pGFP”)
Completely re-suspend the bacterial colonies
into the CaCl2. Don’t leave any clumps
3.
Use a sterile pipette to transfer .25 mL from
the “- DNA” microtube into the “+ DNA”
microtube. Both tubes should now contain
equal quantities of material
4.
Use a sterile pipette to add the contents of
the pGFP tube to the “+ DNA” microtube only
5.
Place both microtubes into the ice bath for
10 minutes
◦ Appropriate sterile pipettes
◦ 4 petri dishes with nutrient agar
◦
◦
◦
◦
- DNA – amp
- DNA + amp
+ DNA – amp
+ DNA + amp
(striped)
(striped)
Transformation:
Chemical and Heat Shock
The CaCl2 in the microtubes serves to chemically shock
the cells, increasing their competence to receive
plasmids. The next step, heat shock, will further
increase their competence.
Timing and temperature control are very important for
successful transformation. Here’s the routine:
1.
Ice bath for 10 minutes
2.
Heat shock at 42oC for 90 seconds
3.
Ice bath for 2 minutes
4.
Add .25 mL of luria recovery broth to each
microtube
5.
Recovery in 37oC bath for 30 minutes
6.
Transfer to culture dishes
For the heat shock you will need to maintain a water
bath at 42oC. It won’t need to be a large water bath,
but you will need to carefully regulate the
temperature. Keep a flask of hot water and a beaker of
room temperature water handy, and add appropriate
quantities of whichever you need to maintain the
desired temperature.
The microtubes will only be in the heat shock bath for
90 seconds, but it is important to keep the temp at 42
When you return the microtubes to the ice bath, add
room temp. water to the bath to drop the temp to
37oC.
Before you put the microtubes in the recovery bath,
remember to add the luria recovery broth!
The microtubes will recover at 37oC for 30 minutes
before you plate the cultures out
Plating the Cell Cultures
Materials:
•
•
•
•
Sterile inoculating loops
Sterile pipettes
4 petri plates, labelled appropriately
2 microcentrifuge tubes (after 30 minute recovery
in 37oC water bath)
• - DNA
• + DNA
• Bleach bath for used pipettes and inoculating loops
• All tools contacting the bacterial cultures must be sanitized in the
bleach bucket. Don’t set used tools down on the lab table. Not
ever. In the bleach bucket. No exceptions. Don’t be the one who
infects another student with E. coli. Seriously. Bleach bucket. I’m
not kidding.
1.
Lay out your petri dishes, make sure that your
– DNA plates are separated from your + DNA
plates
2.
Be careful to transfer the correct cultures to
the correct dishes (- DNA to – DNA, and +
DNA to + DNA), and make sure to use sterile
transfer pipettes and inoculating loops.
3.
Use sterile pipettes to transfer .25 mL of your
cell cultures to the center of the petri dishes.
4.
Use sterile inoculating loops to spread the
cells over the entire plate, first outward from
the center, then laterally, quarter turn, then
laterally again (see pdf p. 13).
Upon successful completion . . .
Stack your petri dishes and tape them
together
Make sure to label your group’s stack of
cultures so you can get the correct one’s back
after they incubate
The dishes will incubate overnight at 37oC
• 37oC, by the way, is your body temperature.
• E. coli is an intestinal bacterium common to
humans, so our optimum temperature is their
optimum temperature
We’ll observe the results the following day.
Before you return to class to observe your
results, you will need to complete your lab
questions (side 1 of the handout only – just
the rationale and the predictions)
Here’s a pretty good video from University of
Pennsylvania that describes the process.
They’ve got better tools than we do, and
they’re doing a follow-up activity that we
won’t be doing, but it’s basically the same lab
https://www.youtube.com/watch?v=iDfEVhePDPM
And a link to Bozeman biology as well
http://www.bozemanscience.com/ap-bio-lab-6-molecular-biology