Bacterial transformation-technical guide - SSERC



Bacterial Transformation using the

Bio-Rad pGLO System

Important Safety Information

The instructions which come with the Bio-Rad pGLO Transformation Kit are written for the North American market and do not conform to the current safety guidelines on the use of micro-organisms issued to Scottish schools and non-advanced FE Colleges. We recommend that the Technical Guide which follows is used to supplement the Technical Guide provided by Bio-Rad.

Aseptic Technique must be followed throughout, i.e. in preparation of materials for classes, by students carrying out the practical work and in disposal.

Preparation for work and all microbiological techniques used should follow the methods described in the Microbiology Techniques Cards issued to all Scottish schools and non-advanced FE Colleges by The Higher Still Development Unit as part of the Higher Still programme. These are now also available from SSERC in electronic format as part of the Microbiology Safety and Techniques section of the SSERC Safety Net CD ROM (.uk). In other areas of the UK, teachers and technicians should seek advice on recommended microbiological procedures from the appropriate authority.

Media should be sterilised by autoclaving at 121(C for 15 minutes.

Cultures should not be incubated at 37(C. They should be incubated at 30(C or below.

The transformed bacteria produced in this experiment are exempt from the ‘Contained Use’ regulations but are subject to the Genetically Modified Organisms (Deliberate Release) Regulations 1992 (as amended 1995, 1997 and 1998). Although E coli K12 used in this kit is a debilitated (weakened) strain and unlikely to survive outside the laboratory, it is essential to make sure that the organisms are not released into the environment. All transformed cultures must be disposed of by autoclaving or in a pressure cooker at a temperature of 121(C for 15 minutes (not by immersing in disinfectant) within seven days of inoculation after which they can be disposed of with normal rubbish.

Introduction

Bacterial Transformation

Bacterial transformation occurs when a bacterial cell takes up a new piece of DNA. We can make use of the process of bacterial transformation to insert a novel gene or genes into an organism.

Lengths of DNA can be manipulated to contain desirable genes (e.g. the gene for human insulin), and then transferred into bacteria. This can be achieved simply by mixing these DNA fragments with bacteria under favourable conditions when some of the DNA will enter some of the bacteria. These newly transformed bacteria may then synthesise the desired protein from the gene.

In this practical, a plasmid (a small circular piece of DNA) is transferred into the bacterium E coli. The E coli cells in the starter culture are sensitive to ampicillin and do not contain the gene for Green Fluorescent Protein (GFP). They, therefore, cannot synthesise GFP and do not fluoresce in the presence of UV light. The pGLO plasmid has been constructed to contain a gene which confers resistance to ampicillin, and part of the operon for arabinose metabolism attached to the gene for Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria (see Fig.1 in ‘Support Information’ at the end of the Student Guide).

Bacterial cells which take up the plasmid become resistant to ampicillin and, if arabinose is present in the medium to ‘switch on’ the arabinose promoter, synthesise Green Fluorescent Protein and ‘glow’ when exposed to UV light. They do not fluoresce if arabinose is not present (see ‘Support Information’ at the end of the Student Guide).

Preparation of materials

A number of materials require to be prepared on advance:

• Agar plates (3 – 7 days)

Three different types of agar plates are used:

LB (Luria Broth): on which both untransformed (starter) and transformed E coli cultures grow. Luria broth/agar is a general nutrient medium.

LB/amp (Luria Broth + ampicillin): on which only E coli that have taken up the plasmid (i.e. transformed and hence resistant to ampicillin) grow. They do not, however, fluoresce as there is no arabinose in the medium.

LB/amp/ara (Luria Broth + ampicillin + arabinose): on which only transformed E coli grow. They do fluoresce as the arabinose in the medium causes the promoter to switch on the gene for GFP.

• Starter cultures and plasmid (2-3 days)

The E coli starter culture and plasmid DNA have been freeze-dried. Both must be rehydrated. The E coli must be grown up on the agar plates. The plasmid DNA, once rehydrated, should be kept refrigerated.

• Solutions and other student materials dispensed

Materials

- Disposal jar containing clear phenolic or Virkon disinfectant at appropriate concentration

- Eye protection

- 1% bleach or hypochlorite solution and paper towels for wiping down working area

- autoclave or pressure cooker

- 1 x 1 dm3 or 2 x 500 cm3 bottles for preparing media

- Measuring cylinder, 500 cm3

- 500 cm3 distilled water

- Incubator at 30(C

- Bio-Rad Transformation Kit (suitable for eight groups of students)

Order from bio-, 166-0003EDU: pGLO Bacterial Transformation Kit. List price £65.00(Feb 2011)

Refill kit, 166-0555EDU. List price £38.00(Feb 2011)

UV lamp, 166-0500EDU. List price £32.00(Feb 2011)

Method

3 – 7 days before practical – Prepare LB Agar

1. Wash your hands and wear a lab coat.

2. Use a paper towel to wipe your bench with 1% bleach.

3. Add 500 cm3 distilled water to a one litre bottle suitable for autoclaving, or 250 cm3 to each of two 500 cm3 bottles.

4. Add the contents of the LB agar packet to the water. (If using two bottles, divide the powder into two equal quantities before adding to the water).

5. Tighten the lid of the bottle (s) then loosen ¼ turn (to allow steam to penetrate during autoclaving).

6. Sterilise the medium by autoclaving at 121(C for 15 minutes.

7. Place in a water bath at 50(C.

8. While the agar is cooling, prepare the arabinose and penicillin, both of which have been freeze-dried for transportation.

9. Using aseptic technique, pipette 3 cm3 Transformation Solution from the kit (or sterile distilled water) to the vial of arabinose.

Note: The arabinose will take about 10 minutes to dissolve. Agitation will help the process.

10. Using a fresh sterile pipette and aseptic technique, add 1 cm3 Transformation Solution or sterile distilled water to the vial containing ampicillin (the antibiotic).

11. On the underside and at the edge, date and label 16 plates ‘LB’, 16 plates ‘LB/amp’ and 8 plates ‘LB/amp/ara’.

12. When the agar has cooled to 50(C, pour 16 LB plates (see Techniques Cards MP 3&4), filling each plate 1/3 – 1/2 full.

13. Check that the ampicillin is fully hydrated and that the agar temperature is not greater than 50(C – ampicillin and arabinose will be destroyed at temperatures greater than this.

14. Using aseptic technique, add all the ampicillin to the remaining molten LB agar.

15. Swirl gently to mix thoroughly.

16. Using aseptic technique, pour 16 LB/amp plates.

17. Add the hydrated arabinose to the remaining molten LB/amp agar.

18. Swirl gently to mix thoroughly.

19. Using aseptic technique, pour 8 LB/amp/ara plates.

20. When the plates have solidified, store upside down in a safe place at room temperature for 2 days.

21. If they are to be stored for longer, place in plastic sleeves in a refrigerator until required.

2 days before practical – rehydrate plasmid DNA and bacteria, inoculate starter plates

1. Wash your hands and wear a lab coat.

2. Use a paper towel to wipe your bench with 1% bleach.

3. Using a fresh sterile pipette and aseptic technique, add 250 (L Transformation Solution or sterile water to the vial of E coli. Ensure cap is replaced.

4. Allow vial to stand at room temperature for 5 minutes.

5. Shake gently to suspend the bacteria in solution.

6. Using aseptic technique, make one streak plate of the E coli starter culture for each group of students. (See techniques Cards SF 4 and ST 3. If you are using sterile plastic loops, do not attempt to flame; where you are instructed to flame the loop between streaks, discard loop into disinfectant then use a fresh loop for the next stage). It is important that your technique produces plates with single colonies growing.

7. Incubate the plates at 30(C for 48 hours. If an incubator is not available, the bacteria will grow at room temperature but will take 3 – 4 days to grow.

8. Using aseptic technique and a fresh sterile pipette, add 250 (L Transformation solution or sterile distilled water to the vial containing the freeze-dried plasmid DNA.

9. Refrigerate till required.

10. Use a paper towel to wipe bench with 1% bleach.

Before practical class

1. Dispense 1 cm3 Transformation Solution into a pink microtube (one per student group).

2. Dispense 1 cm3 LB broth into a red microtube (one per student group).

3. Refrigerate, unless dispensing immediately before class.

4. Set out materials as shown below.

After practical session 1

Note that plates must not be incubated at 37(C as this would contravene the guidelines in the Code of Practice. Instead, cultures should be incubated at 30(C or below. The organisms will take longer to grow at these lower temperatures but final colony numbers and size will be the same.

After practical session 2 (observing and counting colonies)

All transformed organisms must be autoclaved within seven days of inoculation and then disposed of with normal rubbish.

They must not be deliberately released.

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Bacterial Transformation

Teacher & Technical Guide

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