Day 1: Introduction Reading and discussion of considerations Day 2: Transformation: Transform cells and sow plates. Day 3: Collect and analyze results. Observed transformed cells and controls. Day 4: Analyze and interpret results. Discussion and complementary activities.

Sterilization technique

In any microbiological technique it is essential not to introduce contaminating bacteria into the experiment. Since the contaminating bacteria are found everywhere (fingers, table, etc.) it is important to prevent the material from coming into contact with these surfaces. When students are working they should avoid touching the end of the sowing handle, the tip of the pipette and the agar of the plate and should also avoid contact with any contaminated surface. For various reasons, very small quantities and volumes are used in the laboratory, requiring the use of specialized equipment such as analytical scales and micropipettes.

Working with E. coli

The organism used in this process, a strain of E. coli (DH5α), the vector that contains the gene of our interest, and the transformed cells that are formed by combination of the above, are not pathogenic organisms such as E. coli O157:H7, which is known for its involvement in food-poisoning infections. However, the handling of E. coli (DH5α) must be done according to standard microbiological safety standards. Among these we can cite: • Work surfaces are disinfected once a day and each time an accidental spill occurs (with ethanol). • All liquid or solid waste must be disinfected before it is thrown away. • Hands should be washed: or after handling material with recombinant DNA molecules, and or before leaving the laboratory. • All procedures should be performed with caution to minimize aerosol formation. • Micropipettes shall be used, it is forbidden to pipette with the mouth. • It is forbidden to eat, drink, smoke and apply cosmetics in the work area. • Protective glasses and gloves are recommended.

Disinfection and removal of material

With the help of the autoclave, sterilize all used material, then double bag the single-use and discard. Wash well those that are not disposable. Do not throw the agar into the pipe.

This protocol is designed to use an incubator at 37 ºC. The transformation can be done without using the incubator, but then the number of days needed for the colonies to grow will depend on the ambient temperature. The best results are observed with crops of 24-48 hours and with colonies with measures of 1-1.5 mm of diameter. Cooling the plates will significantly reduce the processing efficiency. The optimal growth temperature of E. coli is 37 ºC and lower temperatures will produce a lower growth rate. At 28 ºC, 2 days of incubation are needed to obtain colonies of appropriate size. At 21 ºC, 3 days of incubation are required.

Methodological indications

Transfer of bacterial colonies from the agar plates to the tubes When you take a colony from the culture plates, you may be tempted to take more cells than necessary. It should be remembered that a single colony with a diameter of 1 mm contains millions of cells.

The procedure used to promote foreign DNA collection is called thermal shock. It is important to follow the recommended times. Also important is the sudden change in temperature and the duration of the thermal shock. For optimal results, the tubes with the cell suspension should be removed directly from the ice, placed in a bath at 42 ºC for 50 seconds and immediately put on ice again. For example, if there is no thermal shock, one tenth of transformations will occur, or if the duration of the shock is 90 seconds, half of transformations will be obtained than with 50 seconds.

Adding too much transformation medium to plates is counterproductive as plates cannot absorb excess fluid and seeding will be uneven. The passage of bacterial suspension from tubes to petri dishes should be done with care. The bacteria are deposited at the bottom of the tubes, so the tubes should be shaken. To do this you must take the top of the tube with your thumb and index fingers and you must tap the bottom of the tube with the index finger of the other hand. You should also make sure that the petri dishes are taped once the bacterial suspension has been extended.

Transformation solution

The Ca 2+ ions present in the solution (Cacl2) neutralise the repulsion between the negative charges of the DNA phosphate groups and the cell membrane phospholipids, allowing DNA to enter the cells.

Thermal shock increases cell membrane permeability to DNA. Although it is not known why, the duration of the thermal shock is known to be critical to the process and has been optimised for the type of bacteria and conditions used.

Recovery

The 10 minutes of incubation after the addition of the LB broth allows the cells to grow and express the antibiotic resistance gene, and so the transformed cells survive in the antibiotic plates, which allows to know if the vector has been integrated and is expressing itself. Incubation can be carried out at room temperature or in the incubator at 37 ºC to increase processing efficiency by up to 10 times.

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Team:UAAAN/Experiments