Transformation
Advanced Preparation
- Luria-Bertani (LB) agar: LB agar can be made up at any time. It must
be autoclaved and kept sterile in the refrigerator. Simply follow the directions
on the bottle being sure that you pre-order an ample supply of distilled
water. To avoid serious problems when re-melting the agar later, it is
best to place only 350 ml of agar solution into a 500 ml bottle. Autoclaving
(sterilizing) can be done using a pressure cooker at 15 psi for 20 minutes.
It is simpler but more expensive to buy pre-made, sterile LB agar from
any biological supply company.
- LB plates: Preparing the LB plates is the most demanding part of the
preparation. Since each team requires 6 plates and there are 3 different
types of plates per team and sterile conditions must be maintained, this
needs to be done by the teacher OR students under very close supervision.
This may be an opportunity to work closely with other department members
or your best students as an after school or weekend project.
- Sterilizing plates: Empty glass petri dishes (15 mm X 100 mm) can be
sterilized ahead of time by heating them in an oven at 400ûF for
30 minutes (Caution Hot). Be sure to let them cool several hours before
removing from the oven and handle only with hot gloves. If you can afford
them, buy pre-sterilized plastic petri dishes from any biological supply
company.
- Marking plates: Sterilized petri dishes should be marked on the bottom
close to the edge. Each student team will have 2 plates labeled "LB";
2 plates labeled "LB/AMP"; and 2 plates labeled "LB/AMP/X-gal".
In addition to team sets of dishes, you will need one extra dish per lab
station labeled LB. This dish will be sub-cultured with bacteria 2-3 days
before the lab is done and will be the source of bacteria at that lab station
for each student team throughout the day.
- Pouring plates: Since ampicillin and X-gal are both heat sensitive,
it is essential that you have hot water baths capable of holding temperature
at approximately 60ûC. These can be made using pans or large beakers
over hot plates with variable heat settings. The pre-sterilized agar must
be melted. This can be done with a hot water bath or microwave oven. The
temperature needed is 100ûC and you must LOOSEN THE CAPS on the agar
bottles before heating. When using a microwave, heat at the medium power
setting and remove the caps, completely replacing them with sterile cotton
or tissue. Once melted, the agar must cool to 60ûC (this can take
as long as 20-30 minutes). This temperature can be approximated by feel.
If you can hold both hands around the bottle and not get a burning sensation,
the agar is at the correct temperature. The agar can be placed into the
hot water bath and kept at this temperature for hours. There must be enough
water in the bath to completely cover the agar in the bottles when they
are placed in the bath. Once at this temperature, the Ampicillian and X-gal
solutions can be added to the appropriately marked agar bottles. DO NOT
ADD AMPICILLIN OR X-GAL to agar above 65ûC. When pouring the plates,
place the sterilized, pre-labeled plates along the edges of your lab tables
and lift the lids just enough to get the neck of the agar bottle into the
space between the lid and the bottom of the plate. Pour enough agar (15
ml or less) to BARELY cover the bottom of the plate and replace the lid
immediately. BE SURE THE AGAR IS POURED INTO THE CORRECTLY LABELED PLATE.
To reduce the amount of condensation inside the plate, you can leave the
lid propped at an angle (just the slightest opening) instead of placing
it down flat on the bottom half of the petri dish. After the agar in the
plates cools and solidifies, close the plates, turn them over and stack
them. They can be stored in plastic bags in the refrigerator until lab
day. Left over agar labeled LB can be saved in the refrigerator and re-melted
for later use. Agar labeled AMP or AMP/X-gal CAN NOT be saved for future
use. Re-melting this agar would destroy the AMP and X-gal molecules. Once
you begin this preparation, you cannot leave until all the AMP, X-gal plates
are poured. Be sure to plan plenty of time for this portion of the preparation;
several hours are usually required. You might consider pouring LB plates
one day and then the AMP and AMP/X-gal plates on another day.
- Sub-culturing bacteria: Two or three days before the activity is planned,
you need to take the extra LB plates poured (the ones you determined would
be the source of bacteria for each lab station) and streak them with E.
coli bacteria.
- Transformation Kit: The transformation kits need to be ordered 6 weeks
in advance of their planned use. They can be ordered form Carolina Biological
Supply Company catalog # F6-21-1146. Each kit comes with enough supplies
and consumables for SIX TEAMS; you need to divide your classes accordingly.
If the LB broth or calcium chloride comes in large containers, you will
have to transfer these to 1.5 ml microcentrifuge tubes. Since most students
have poor technique and don't measure very accurately with dropping pipets,
you should make a double set of these tubes. This will prevent your having
to try to transfer into these tubes during a class period.
- Sterile technique: You should explain to your students the meaning
of "sterile" and review those parts of the procedure that require
using sterile technique. The procedure for this lab is written to save
materials. In doing this, some sterile technique is sacrificed. If you
have ample materials, instruct your students to discard all pre-sterilized
pipets and loops after one use. You may choose to use a glass spreading
rod for spreading bacteria rather than the sterile loop technique in the
procedure. If so, be sure to place the ethyl alcohol (used to flame the
glass rod) in petri dishes or beakers and keep it and the glass spreading
rod away from the open flame of the Bunsen burner. You will have to demonstrate
how to flame and use the glass spreading rod.
- Preparation time: Estimated time for making LB agar and marking plates
is 11/2 hours. Estimated time for sterilizing plates is 1 hour. Estimated
time for pouring plates is 2-3 hours. Estimated time for sub-culturing
bacteria is 10 minutes. Total estimated preparation time for 5 classes
is 6 hours.
Introduction
This exercise allows students to learn the basic technique of inserting
a piece of foreign DNA into a host organism. They will perform a transformation
upon the bacteria Escherichia coli by inserting a plasmid containing two
genes not normally found in the bacteria. By using pre-selected growth
media, they can distinguish the bacteria cells that were transformed. This
technique gives the students insight about one basic method used in genetic
engineering.
Student Objectives
- The student will define transformation.
- The student will transform a bacterium.
- The student will complete sterile technique.
- The student will interpret and analyze experimental results using comparisons
with controls.
- The student will design an experiment using transformation technology.
- The student will calculate transformation efficiency.
Class Time Needed
Four to five 50-55 minute periods are required to do this activity.
- The first day is for transforming and plating bacteria. Some teachers
may wish to spend 1 day just reviewing and practicing the procedure and
the second day actually performing the lab.
- The second day is for discussions, predictions and gathering the data.
- The third day is for analysis of results and calculation of efficiency.
- The fourth day is for designing another transformation experiment and
discussing that design with the entire class.
Materials
- Sterile plastic pipet (4)
- Sterile plastic loop (4)
- E.coli culture
- pUC 19 plasmid
- 50 mM Calcium chloride
- LB plates (2)
- LB/AMP plates (2)
- LB/AMP/X-gal plates (2)
- 2 sterile culture tubes (15 ml )
- Luria broth
- Test tube holder
- Ice bucket with crushed ice
- 42ûC water bath
- Black permanent marker
- OPTIONAL - INCUBATOR
- 10% bleach solution
- E. coli sub-cultured agar plates
Recipes for consumables
10% Bleach: Add 10 ml of bleach (household variety) to 90 ml of tap
water.
50mM Calcium chloride: Add 50 ml of 1 M Calcium chloride to 950 ml of
distilled water. Pre-rinse sterile filter with distilled water. Pass the
Calcium chloride solution through the pre-rinsed filter. Transfer Calcium
chloride to small, sterile tubes and store refrigerated until used.
Other consumables are mixed according to directions on the stock bottles
or are provided in the correct concentrations by the vendor.
Procedure
The most important thing for students to understand is the idea of STERILE
TECHNIQUE. If they think that they have contaminated a pipet or loop, it
is essential that they discard that tool and get a new one. You should
have an ample supply of sterile pipets and loops at each station. These
can be placed in an empty beaker or jar.
The transfer of plasmid DNA from its stock tube to the pUC+ tube is
crucial. Students must look carefully at the loop to see if there is a
film of plasmid solution across the ring. This is similar to seeing a soapy
film across a wire ring for blowing soap bubbles. If you prefer, you can
have your students transfer 10 µl of plasmid DNA with the micropipet.
This will require extra materials and preparation if you choose this method.
It is essential that the students follow the directions regarding time.
The pUC tubes are placed on ice for 15 minutes; the heat shock, for exactly
11/2 minutes; and the recovery in ice, for 2-3 minutes. These times are
designed to maximize the number of transformants. The most critical part
is getting the shocked tubes BACK IN ICE IMMEDIATELY.
Transferring bacterial suspension from the pUC tubes to the petri dishes
requires some care. The bacteria will settle to the bottom, so be sure
that students stir the suspension with the pipet before drawing it up.
Also, make sure that they don't open the petri dish to the air when placing
the bacteria on the agar or when spreading the bacteria across the agar
surface.
Some teachers like to DRY RUN the procedure first to give ample time
to explain sterile techniques, practice using the pipets and loops and
streaking and spreading bacteria on the agar's surface without completely
removing the petri dish top. You will have to decide what is best for your
students, based upon their lab experience and familiarity with these techniques.
Disposal
- All loops and pipets can be placed in a 10% bleach solution for sterilization.
It might be a good idea to have a shallow pan of this solution at every
lab station. No matter what you choose, all used loops and pipets should
be collected for sterilization.
- Sterilize petri dishes by covering the agar with the 10% bleach solution.
Let it stand for 10 minutes or more. Once sterilized, the agar can be placed
in double zip lock bags and placed in the garbage. Arrange to have your
custodian pick these bags up the day you clean up.