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.
1. 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.
2. 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.
3. 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.

1. 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.
2. The second day is for discussions, predictions and gathering the data.
3. The third day is for analysis of results and calculation of efficiency.
4. The fourth day is for designing another transformation experiment and discussing that design with the entire class.

Materials

1. Sterile plastic pipet (4)
2. Sterile plastic loop (4)
3. E.coli culture
4. pUC 19 plasmid
5. 50 mM Calcium chloride
6. LB plates (2)
7. LB/AMP plates (2)
8. LB/AMP/X-gal plates (2)
9. 2 sterile culture tubes (15 ml )
10. Luria broth
11. Test tube holder
12. Ice bucket with crushed ice
13. 42ûC water bath
14. Black permanent marker
15. OPTIONAL - INCUBATOR
16. 10% bleach solution
17. 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.