Mycobacterial pathogenic strategies remain poorly understood the slow growth rate of most species. The impenetrable nature of the cell wall and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video, we will demonstrate the technique of electroporation, which involves subjecting cells to a brief and high electrical impulse, which allows the entry of DNA.
It is the most widely used method for introducing DNA in mycobacterial cells. Hi, I'm Ron Good from the laboratory of Tanya Parish from the Center of Infectious Disease at the Bats and the London School of Medicine and Dentistry. Today we will show you a procedure for electro operating microbacteria.
In our lab, we study microbacterium tuberculosis, but because it is highly pathogenic and requires a C3 containment facilities, we will show you this technique in the non-pathogenic microbacterium m Mcma. So let's electro ate some bacteria. To ate mycobacteria, we need to make competent cells.
It is important to remember that pathogenic mycobacteria represent an important biohazard. Therefore, all culture and genetic manipulation must be carried out in an appropriate containment. Facilities inside a class one safety cabinet Risk assessment must form the first part of any experiment with pathogenic mycobacteria.
To make these, we start with the culture that has been maintained in the laboratory on solid mediums such as lemko, auger inoculate, five mils Lemko containing tween broth with a loop full of mycobacteria. Since many mycobacteria are relatively slow growing organisms is extremely important to maintain good aseptic technique to prevent contamination of these cultures with bacteria of fungus that can rapidly outgrow the mycobacteria. It is often wise to set up duplicate cultures in case one becomes contaminated.
Mycobacteria cells have a tendency to clump together in culture due to their thick waxy coat. Vortexing and the addition of non ionic detergent such as tween 82 medium reduces this clumping and provides a more homogeneous suspension of cells. After inoculation, incubate the culture at 37 degrees Celsius with shaking at a hundred RPMs overnight.
After the starter culture has grown, inoculate a large scale culture about a hundred mils of lemko tw in a 250 mil conical flask with a one to 100 dilution of the overnight culture. Continue incubating at 37 degrees Celsius with shaking for about 16 to 24 hours at which time the optical density can be checked with the spectrophotometer. If it reads 0.8 to one cells are in their optimal log phase of growth.
Afterwards, incubate the cells on ice for 1.5 hours before harvesting, which will help increase transformation efficiency by fourfold.Longer. Incubations on ice result in reduced efficiency. Next, harvest the cells by pouring the cultures into large centrifugation tubes.
Spin at 300 Gs for 10 minutes after centrifugation wash the cells three times in ice cold, 10%glycerol reduce the volume each time, such that for a culture volume of a hundred meals, 20 meals is used for the first wash, then 10 mils and finally five mils for the last wash. Following the washes, resus suspend the cells in one to 10 to one to a hundred of the original volume of ice cold. 10%glycerol at this stage, cells can be aliquot, frozen and stored at minus 70 degrees Celsius for future use.
The transformation efficiency often increases after freezing the cells. Before using these competent cells, it should be thawed, harvested by centrifugation and resuspended in fresh, 10%glycerol prior to use. In order to electro parade, ems mag an electro, which is an apparatus that can deliver a high voltage pulse of 2.5.
Kilovolts is used. Bacteria and DNA are placed in electroporation, cuvettes. And although the gap or path length may be the same in different cuvettes, they vary with respect to the volume that they can hold.
We routinely use 200 microliters of cells in a 0.2 centimeter qve to ate the cells. First, prepare the DNA sample such that it is free from salts and other contaminants. This is important to minimize the chances of arcing during electroporation.
Prior to poring bacteria, make sure that your DNA samples are of high purity. The two 60 to two 80 absorbent ratio should be close to 1.8. Add 0.5 to five micrograms of the salt-free DNA in no more than five microliters volume to 200 microliters microbacterial suspension, A control electroporation with no DNA to check for the frequency of antibiotic resistance mutants can be included.
Then leave the mix on ice for 10 minutes. This will reduce the presence of life cells in the sample and salts in the DNA solution, which will minimize arcing after the ice incubation, redistribute the cells thoroughly with a pipette or vortex before the high voltage pulse to resuspend the cells and ensure mixing of the DNA. Any clumping of the cells will lead to arcing and reduce transformation efficiency.
Next, transfer the mix to a pre chilled 0.2 centimeter electrode gab qve. Again, take care not to introduce any bubbles into the mix. Also to reduce arcing.
Make sure that the outside of the qve is completely dry before placing it in the pulse chamber. After drying the qve thoroughly and with the pulse controller resistance set to a thousand ohms, place the qve in the electroporation chamber. Then after setting the voltage to 2.5 kilovolts, apply the pulse until a beep is hurt, indicating that the capacitor in the QVE has discharged.
A time constant of greater than 10 will indicate a successful electroporation. Unsuccessful electroporation are usually indicated by arcing, which causes a spark, which can often be avoided. By changing the resistance on the pulse controller, put the Q vet back on ice for 10 minutes.
After incubation, transfer the cell suspension to a sterile universal bottle containing five mils of lemko TW broth. It is important to dilute the cells at least tenfold immediately after the pulse to help the cells recover and increase survival of the transformants. Next, incubate the cells at 37 degrees Celsius for two to three hours, and then they're ready to be plated after the incubation, harvest the cells by centrifugation at 3000 Gs for 10 minutes.
Following centrifugation, make suitable dilution of the suspension in lemko broth such that each plate will get 30 to 300 colonies. It is important to ensure that resistant colonies have arisen from a single cell, so be sure cells are thoroughly resuspended before plating. If the cells are not diluted, it can be very difficult to visualize truly resistant colonies against a background Lawn of sensitive cells now plate out cells on lemko auger with the appropriate antibiotic.
They incubate the plates at 37 degrees Celsius until colonies become visible. This will take three to five days after the colonies have grown. Count the transformants.
To calculate transformation efficiency. Several factors affect the efficiency of transformation. These include the growth phase of cells when harvested electroporation media, and the field strength and time constant of the delivered pulse.
The efficiency of the electroporation technique also depends on the choice of DNA for transformation, and sometimes on the choice of the selectable marker. We routinely obtain efficiencies of 10 to the fifth, to 10 to the sixth colonies per microgram, DNA, using hygromycin or streptomycin resistance in ems magma. Now to grow out the transformants streak out the electroporated mycobacteria onto solid medium plus selection.
Antibiotic incubate at 37 degrees for two to three days. Following the two to three day incubation, you can analyze transformants as required for most applications, three transformants should be streaked out and analyzed. It is important to check the identity of extra chromosomal plasmid after transformation.
Since deletions and rearrangements are common, I've just shown you how to electrolyte mycobacterium s magma. If you're going to do this procedure with pathogenic microbacteria, remember to use appropriate safety measures. So that's it.
Thanks for watching and good luck with your experiments.
