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Lab 6A Bacterial Transformation
Introduction:
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 avirus 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 acell by directly taking up the DNA. This lab uses transformation to insert a specific geneinto 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 plasmidvectors also carry genes for antibiotic resistance, transformed cells containing plasmidsthat carry the foreign DNA of interest in addition to the antibiotic resistance gene can beeasily selected from other cells that do not carry the gene for antibiotic resistance. Theyare usually extrachromosomal. This means they exist separately from the chromosome.Some plasmids replicate only when the bacterial chromosome replicates, and usuallyexist 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. Itis a common inhabitant of the human colon and can easily be grown in suspension culturein a nutrient medium such as Luria broth, or in a petri dish of Luria broth mixed withagar, or nutrient agar. The single circular chromosome of E. coli contains about fivemillion DNA base pairs, only one-six hundredth of the haploid amount of DNA in ahuman 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 hastransformed the bacteria so that they are resistant to the antibiotics.
Materials:
The materials needed in this lab were two Luria agar plates, two Luria agar plates withampicillin, two 15mL tubes, one inoculating loop, one bacterial spreader, several sterilemicropipettes, calcium chloride, Luria broth, pAMP solution, a Bunsen burner, hotplate,ice, and a water bath.
 
Methods:
Mark one of the sterile 15mL tubes "+" and the other "-", the plus tube obviously havingthe plasmid added to it while the other tube does not receive any. Using a sterilemicropipette, add 250 microliters of ice cold 0.05M CaCl
2
to each tube. Transfer a large3mm colony of E. coli from the starter plate to each of the tubes using a sterileinoculating loop. Try to get the same amount of bacteria into each tube. Be careful not totransfer any agar. Vigorously tap the loop against the wall of the tube to dislodge the cellmass. Mix the suspension by repeatedly drawing in and emptying a sterile micropipettewith the suspension. Add ten microliters of pAMP solution directly into the cellsuspension in the tube labeled with a plus sign. Mix by tapping the tube. This solutioncontains the antibiotic resistance plasmid. Keep both of these tubes in ice for about 15minutes. 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 isessential that cells be given a sharp and distinct shock; so take the tubes directly from theice to the water bath. Immediately return the tubes to the ice after ninety seconds. Use asterile micropipette to add 250 microliters of Luria broth to each tube. Mix by tapping thetube. 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 cellsshould be placed. Immediately spread the cells using a sterile spreading rod. This can beaccomplished by running the rod through the Bunsen burner and allowing to cool bytouching it to the agar on the part of the dish away from the bacteria. Spread the cells andonce 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.
Data:
Luria agar +LawnLuria agar -LawnLuria agar with ampicillin +None visibleLuria agar with ampicillin -None visible 
 
Total mass of plasmid used0.05 microlitersTotal volume of suspension510 microlitersFraction of cell suspension put on the plate0.1960784314Total mass of plasmid in fraction0.0098039216 Number of colonies/ microliter of plasmidNone visible
Questions:
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 eachcolony 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 thenutrient. They both had lawns of bacteria colonies on them because the E. coli grownaturally without the presence of the antibiotic.
b. LB/Amp- and LB/Amp+
= The LB on the ampicillin agar with the addition of thegene for transforming the plasmids of the E. coli should be able to survive, maintainingthe characteristics of the gene present in their DNA. The LB should not have survived onthe agar with ampicillin because this is how it would occur in nature, and there is no genein the bacterium to help resist the antibiotic.
c. LB/Amp+ and LB+
= These two should both have growth. They both were exposed tothe 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 lessgrowth on the ampicillin plate because not all of the bacteria cells could havetransformed; this would have only occurred under optimal conditions.
Determine the total mass of pAMP used.
(Ten microliters were used at a concentrationof .005 microgram/microliter) There were .05 microliters used.
Calculate the total volume of cell suspension prepared.
There was a total of 510microliters used.
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Dyslexia: In Table 6.1, 2nd down on the left, it should be 9,416. The other values are correct.

reply03 / 12 / 2011
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