|Lab 6A Bacterial Transformation|
Genes are transferred between bacteria by way of conjugation, transduction, or transformation. Conjugation takes place when the genetic material is transferred from one bacterium to another of a different mating type. Transduction requires the presence of a virus to act as a vector, or a carrier to transfer small pieces of the DNA from one bacterium to another. Transformation involves the transfer of genetic information into a cell by directly taking up the DNA. This lab uses transformation to insert a specific gene into a plasmid so that the cell takes on those characteristics for which the gene codes.
Plasmids are small rings of DNA that do carry genetic information. They can transfer genes, like genes for antibiotic resistance, which can occur naturally within them, or plasmids can act as carriers or vectors for introducing foreign DNA from other bacteria, plasmids, or even eukaryotes into recipient bacterial cells. Restriction endonucleases can be used to cut and insert pieces of foreign DNA into the plasmid vectors. If these plasmid vectors also carry genes for antibiotic resistance, transformed cells containing plasmids that carry the foreign DNA of interest in addition to the antibiotic resistance gene can be easily selected from other cells that do not carry the gene for antibiotic resistance. They are usually extrachromosomal. This means they exist separately from the chromosome. Some plasmids replicate only when the bacterial chromosome replicates, and usually exist only as single copies within the bacterial cell, but still others replicate on their own, autonomously. There can be anywhere from ten to two hundred copies within a single bacterial cell. There are specific plasmids called R plasmids that carry genes for resistance to antibiotics such as ampicillin, kanamycin, or tetracycline.
The bacterium Escherichia coli, or E. coli, is an ideal organism for the molecular geneticists to manipulate and has been used extensively in recombinant DNA research. It is a common inhabitant of the human colon and can easily be grown in suspension culture in a nutrient medium such as Luria broth, or in a petri dish of Luria broth mixed with agar, or nutrient agar. The single circular chromosome of E. coli contains about five million DNA base pairs, only one-six hundredth of the haploid amount of DNA in a human cell. Also, the E. coli cell may contain small plasmids, discussed earlier. The plasmids are broken up with calcium chloride, and the wanted gene is inserted and the bacteria can be grown on the nutrient or with an antibiotic to see if the gene has transformed the bacteria so that they are resistant to the antibiotics.
The materials needed in this lab were two Luria agar plates, two Luria agar plates with ampicillin, two 15mL tubes, one inoculating loop, one bacterial spreader, several sterile micropipettes, calcium chloride, Luria broth, pAMP solution, a Bunsen burner, hotplate, ice, and a water bath.
Mark one of the sterile 15mL tubes “+” and the other “-“, the plus tube obviously having the plasmid added to it while the other tube does not receive any. Using a sterile micropipette, add 250 microliters of ice cold 0.05M CaCl2 to each tube. Transfer a large 3mm colony of E. coli from the starter plate to each of the tubes using a sterile inoculating loop. Try to get the same amount of bacteria into each tube. Be careful not to transfer any agar. Vigorously tap the loop against the wall of the tube to dislodge the cell mass. Mix the suspension by repeatedly drawing in and emptying a sterile micropipette with the suspension. Add ten microliters of pAMP solution directly into the cell suspension in the tube labeled with a plus sign. Mix by tapping the tube. This solution contains the antibiotic resistance plasmid. Keep both of these tubes in ice for about 15 minutes. While the tubes are on ice, obtain two LB agar plates and two LB/Amp agar plates. Label each plate on the bottom as follows: one LB agar plate “LB+” and the other “LB-.” Label one LB/Amp plate “LB/Amp+” and the other plate “LB-.” A brief pulse of heat facilitates entry of foreign DNA into the E. coli cells. Heat-shock cells in both the + and – tubes by holding in a water bath of 42 degrees Celsius for ninety seconds. It is essential that cells be given a sharp and distinct shock; so take the tubes directly from the ice to the water bath. Immediately return the tubes to the ice after ninety seconds. Use a sterile micropipette to add 250 microliters of Luria broth to each tube. Mix by tapping the tube. Any transformed cells are now resistant to ampicillin because they contain the gene. Place 100 microliters of the + cells on the LB+ plate and on the LB- plate, the other cells should be placed. Immediately spread the cells using a sterile spreading rod. This can be accomplished by running the rod through the Bunsen burner and allowing to cool by touching it to the agar on the part of the dish away from the bacteria. Spread the cells and once again run the rod through the fire to sterilize the rod. Allow the plates to set for several minutes, then tape the plates together and incubate inverted overnight.
|Luria agar +||Lawn|
|Luria agar –||Lawn|
|Luria agar with ampicillin +||None visible|
|Luria agar with ampicillin –||None visible|
|Total mass of plasmid used||0.05 microliters|
|Total volume of suspension||510 microliters|
|Fraction of cell suspension put on the plate||0.1960784314|
|Total mass of plasmid in fraction||0.0098039216|
|Number of colonies/ microliter of plasmid||None visible|
Observe the colonies through the bottom of the culture plate. Do not open the plates. Count the number of individual colonies; use a permanent marker to mark each colony as it is counted. If cell growth is too dense to count individual colonies, record “lawn.”
LB+ (positive control) – lawn. LB- (positive control) – lawn.
LB/Amp + (experimental) – none visible. LB/Amp- (negative control) – none visible.
Compare and contrast the number of colonies on each of the following pairs of plates. What does each pair of results tell you about the experiment?
a. LB+ and LB- = These are two controls without the presence of ampicillin in the nutrient. They both had lawns of bacteria colonies on them because the E. coli grow naturally without the presence of the antibiotic.
b. LB/Amp- and LB/Amp+ = The LB on the ampicillin agar with the addition of the gene for transforming the plasmids of the E. coli should be able to survive, maintaining the characteristics of the gene present in their DNA. The LB should not have survived on the agar with ampicillin because this is how it would occur in nature, and there is no gene in the bacterium to help resist the antibiotic.
c. LB/Amp+ and LB+ = These two should both have growth. They both were exposed to the gene that protects E. coli bacteria against ampicillin and so they both should survive because it shouldn’t matter if the ampicillin is present or not. There is probably less growth on the ampicillin plate because not all of the bacteria cells could have transformed; this would have only occurred under optimal conditions.
Determine the total mass of pAMP used. (Ten microliters were used at a concentration of .005 microgram/microliter) There were .05 microliters used.
Calculate the total volume of cell suspension prepared. There was a total of 510 microliters used.
Now calculate the fraction of the total cell suspension that was spread on the plate. The fraction of the total cell suspension that was spread was 0.1960784314.
Determine the mass of pAMP in the cell suspension. The mass of the pAMP in the cell suspension was 0.0098039216.
Determine the number of colonies per microliter of plasmid. Express the number in scientific notation. There were none visible.
There were many possibilities for error in this lab. The amounts of the different solutions could have been mixed up of misread. Also, there were many lumps in the agar poured into the plates. The spreading rod that was used to spread the bacteria onto the agar could have been too hot when it was used to spread, and killed the bacteria, or while the lid of the petri dish was off, some other contamination from the room could have infected the bacteria causing different results. Finally, another place of error could have been in setting of the bacteria on the plates.
This lab shows the transformation of E. coli bacteria cells can affect the resistance those bacteria cells have to the antibiotic ampicillin. The transformation takes place when the plasmids of the bacteria are opened to taking in foreign DNA by the addition of calcium chloride, and they were finally accepted with the help of heat shock. The results show that if the bacteria cells were transformed, the cells could grow on the agar plates with ampicillin, now having a gene for resistance to that antibiotic.