Our research explores the regulation of mitochondrial protein synthesis and its connection to inner membrane assembly, crucial for oxidative phosphorylation. Using Saccharomyces cerevisiae as a model, we studied the molecular mechanism of mitochondrial function, leveraging its suitability for detailed molecular and genetic investigations. Many technologies are used for research in our field, like RNA deep sequencing, multiomic technologies, cryo-electron microscopy, high-velocity bombardment to transform yeast mitochondria among others.
We have made important contributions to understand the role and mechanisms of action of many proteins involved in mitochondrial translation regulation, and on respiratory complexes III and IV assembly. This is a case for proteins like MSS51, PET309, PET54, mS38, cvp3, cvp6, among others. This protocol is an indispensable tool for understanding how mitoribosomes find the StAR in non-mitochondrial mRNAs.
It has been the key to understanding the role of the specific nucleotides of mitochondrial mRNAs in translation regulation. These are some examples of what these technique can address. The only available resource to proteinase mitochondrial sequences and to use translation of the in the mitochondrial DNA is our protocol.
High velocity bombardment transformation of these mitochondria. This method has provided invaluable information regarding translation and respiratory complexes assembly that otherwise we could not obtain. To begin, add 1.5 milliliters of 70%ethanol to 30 milligrams of 0.7 micrometer tungsten particles in a micro tube.
Centrifuge the mixture at 13, 200 G for 15 minutes. Add 1.5 milliliters of sterile water to the particles then vortex and centrifuge again. After discarding the supernatant, add 500 microliters of sterile 50%glycerol to the tungsten particles.
To coat the particles, first add five micrograms of a two micron plasmid and 15 micrograms of a bacterial plasmid containing the mitochondrial sequence into a micro tube placed on ice. Add 100 microliters of the tungsten particle suspension to the tube and vortex. Then add four microliters of one molar spermidine and vortex again.
Now pipette 100 microliters of ice cold 2.5 molar calcium chloride into the tube. After briefly vortexing the suspension incubated on ice for 10 minutes. Centrifuge the tungsten particles at 13, 200 G for 30 seconds at four degrees Celsius.
Then wash the particles in 200 microliters of 100%ethanol at minus 20 degrees Celsius. Finally, re-suspend the tungsten particles in 60 to 70 microliters of room temperature 100%ethanol. To prepare the biolistic materials, place six sterilized macro carrier holders on a sterile glass plate.
Use a pair of sterile forceps to insert the macro carriers into each of the six macro carrier holders. Pipette 10 microliters of the DNA coated tungsten particles onto each macro carrier surface and evenly distribute over the entire surface with the pipette tip. Inoculate the roh zero yeast receptor strain in two milliliters of YPR medium.
Grow the culture overnight at 30 degrees Celsius in a rotating wheel. The next day, transfer 300 microliters of the culture into a tube containing 30 milliliters of fresh YPR medium. Place the culture on a rotary shaker for two nights at 30 degrees Celsius and 200 revolutions per minute until the culture is saturated.
Centrifuge the yeast cells at 600 G for five minutes at room temperature. Re-suspend the cells in 600 microliters of YPD medium after discarding the supernatant. Now use a sterile glass handle to spread 100 microliters of the yeast cell suspension on a solid bombardment medium plate.
After spreading all the cultures, incubate the plates at room temperature for one to three hours before bombardment. To begin, add 70%ethanol to the chamber of the biolistic particle delivery system. After 15 minutes, wipe the ethanol with a clean paper towel.
Open the helium tank valves to set a pressure of 2000 pounds per square inch. Next, use a pair of sterile forceps to place a rupture disc on the retaining cap and screw the retaining cap on the acceleration tube. Use a pair of forceps to place a macro carrier facing down on the macro carrier holder.
Then screw the macro carrier lid on top of the macro carrier. Place the micro carrier launch system inside the vacuum chamber at the fifth level from the bottom. Then place a plate containing the roh zero strain lawn on the second level from the bottom of the chamber.
Put the vacuum vent hold switch in the vacuum position. When the vacuum reaches 20 to 21 inches of mercury, change the vacuum vent hold switch to the hold position. Press the fire switch to shoot the tungsten particles.
When the pressure reaches 1, 300 pounds per square inch, the rupture disc will break making a pop sound. Immediately after the rupture disc breaks, change the vacuum vent hold switch to the vent position to release the fire switch and the vacuum. Use sterile forceps to carefully remove any debris from the rupture disc present on the surface of the plate.
Then cover the plate with its lid. To begin, incubate the biolistic bombardment plates of yeast at 30 degrees Celsius for five to seven nights. Inoculate the tester strain of the opposite mating type in two milliliters of YPD medium overnight.
The next day, transfer 150 microliters of the tester strain culture on a YPD plate. With a sterile glass handle, spread the tester evenly on the plate surface and let it air dry. Use velvet replicating pads and a replicator stamp to make a master plate replica of the transformed plasmid auxotrophic marker on dropout medium plate.
Then replicate on the YPD plate containing the tester's lawn and incubate. After 48 hours, replicate the plate containing the tester on a selective medium to detect the presence of the mitochondrial gene of interest. Compare the master plate and the corresponding colonies to identify the positive colonies.
Streak the positive colonies from the master plate onto a second selective dropout plate. The tester lawn and the transformant cells mated and resulted infused mitochondria. Purified positive synthetic rho minus colonies were obtained from negative URA plates.
Some positive synthetic roh minus colonies lost the mitochondrial construct. To begin, inoculate two tubes containing three milliliters of YPD media with the synthetic roh minus cells and the accepter yeast cells. The next day, transfer 750 microliters of the donor strain and 250 microliters of the receptor strain into a sterile micro tube.
After centrifuging the mixture for one minute at 13, 200 G, transfer a drop of the cell palate onto a YPD plate and incubate. Check the culture under a light microscope for the presence of schmooze. If schmooze are present, re-suspend a small amount of the cell mass in two milliliters of YPD medium.
Incubate the culture for two hours at 30 degrees Celsius in a rotating wheel. Vortex the mixture well. Then dilute 10 microliters of the cell suspension in 990 microliters of sterile water.
Spread 100 microliters of the diluted culture on a dropout medium that facilitates the growth of only the accepter strain. Replicate the resulting colonies on the necessary selective media. After identifying positive colonies re-streak them on the same selective media to purify the haploid strain carrying the mitochondrial DNA of interest.
