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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.
Use a sterile micropipette to add 250 microliters of Luria broth to each tube. Any transformed cells are now resistant to ampicillin because they contain the gene. Allow the plates to set for several minutes." A brief pulse of heat facilitates entry of foreign DNA into the E. Be careful not to transfer any agar. the other cells should be placed. Transfer a large 3mm colony of E.plate. Mix the suspension by repeatedly drawing in and emptying a sterile micropipette with the suspension. While the tubes are on ice. coli from the starter plate to each of the tubes using a sterile inoculating loop. This solution contains the antibiotic resistance plasmid.05M CaCl2 to each tube. Vigorously tap the loop against the wall of the tube to dislodge the cell mass. Mix by tapping the tube. Using a sterile micropipette. add 250 microliters of ice cold 0. 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. Add ten microliters of pAMP solution directly into the cell suspension in the tube labeled with a plus sign. Spread the cells and once again run the rod through the fire to sterilize the rod. Immediately spread the cells using a sterile spreading rod. Place 100 microliters of the + cells on the LB+ plate and on the LB. It is essential that cells be given a sharp and distinct shock. so take the tubes directly from the ice to the water bath." Label one LB/Amp plate "LB/Amp+" and the other plate "LB-.Methods: Mark one of the sterile 15mL tubes "+" and the other "-". Keep both of these tubes in ice for about 15 minutes. Data: Luria agar + Luria agar Luria agar with ampicillin + Luria agar with ampicillin Lawn Lawn None visible None visible . Mix by tapping the tube. then tape the plates together and incubate inverted overnight. Try to get the same amount of bacteria into each tube. Heat-shock cells in both the + and – tubes by holding in a water bath of 42 degrees Celsius for ninety seconds. Label each plate on the bottom as follows: one LB agar plate "LB+" and the other "LB-. coli cells. Immediately return the tubes to the ice after ninety seconds. obtain two LB agar plates and two LB/Amp agar plates. the plus tube obviously having the plasmid added to it while the other tube does not receive any.
There is probably less growth on the ampicillin plate because not all of the bacteria cells could have transformed. What does each pair of results tell you about the experiment? a. LB/Amp. Compare and contrast the number of colonies on each of the following pairs of plates. There was a total of 510 microliters used. record "lawn. LB/Amp. Count the number of individual colonies. If cell growth is too dense to count individual colonies. and there is no gene in the bacterium to help resist the antibiotic. Calculate the total volume of cell suspension prepared.05 microliters 510 microliters 0.0098039216 None visible Questions: Observe the colonies through the bottom of the culture plate.Total mass of plasmid used Total volume of suspension Fraction of cell suspension put on the plate Total mass of plasmid in fraction Number of colonies/ microliter of plasmid 0.(positive control) – lawn. The LB should not have survived on the agar with ampicillin because this is how it would occur in nature. .= These are two controls without the presence of ampicillin in the nutrient. c.and LB/Amp+ = The LB on the ampicillin agar with the addition of the gene for transforming the plasmids of the E." LB+ (positive control) – lawn.005 microgram/microliter) There were . use a permanent marker to mark each colony as it is counted. Determine the total mass of pAMP used. LB+ and LB. They both were exposed to the gene that protects E. (Ten microliters were used at a concentration of . Do not open the plates.(negative control) – none visible. maintaining the characteristics of the gene present in their DNA. coli grow naturally without the presence of the antibiotic. They both had lawns of bacteria colonies on them because the E. LB/Amp + (experimental) – none visible. b. LB. this would have only occurred under optimal conditions. coli should be able to survive. coli bacteria against ampicillin and so they both should survive because it shouldn’t matter if the ampicillin is present or not. LB/Amp+ and LB+ = These two should both have growth.05 microliters used.1960784314 0.
The transformation takes place when the plasmids of the bacteria are opened to taking in foreign DNA by the addition of calcium chloride. If there is a fourth letter. Finally. it stands for the strain of the organism. The resulting product is usually fragments of DNA of various lengths. The restriction enzymes are named according to a system of nomenclature. The mass of the pAMP in the cell suspension was 0. The fraction of the total cell suspension that was spread was 0.DNA Fingerprinting Introduction: Restriction enzymes are endonucleases that actually cut the phosphodiester bonds on the sides of deoxyribonucleic acid. Determine the mass of pAMP in the cell suspension. The results show that if the bacteria cells were transformed. and killed the bacteria. The next two letters come from the species name.1960784314. This is the reason for the complementary ends. The spreading rod that was used to spread the bacteria onto the agar could have been too hot when it was used to spread. if there are Roman numerals. In humans.0098039216. Express the number in scientific notation. no two individuals have the exact same restriction enzyme pattern in the DNA except for identical twins. or while the lid of the petri dish was off. The amounts of the different solutions could have been mixed up of misread. Error Analysis: There were many possibilities for error in this lab. another place of error could have been in setting of the bacteria on the plates. These endonucleases recognize specific DNA sequences in double-stranded DNA. By using the same restriction enzyme to cut DNA from different organisms. Lab 6B . The first letter represents the genus name of the organism. Conclusion: This lab shows the transformation of E. coli bacteria cells can affect the resistance those bacteria cells have to the antibiotic ampicillin. The endonucleases then digest the DNA at these sites. Some restriction enzymes cut cleanly through the DNA double helix while some produce uneven or sticky ends. In DNA.Now calculate the fraction of the total cell suspension that was spread on the plate. which is usually a four to six base pair sequence of nucleotides. Finally. the antiparallel strands are difficult to deal with considering the restriction enzymes cut from opposite directions. it represents whether that particular . Also. Determine the number of colonies per microliter of plasmid. some other contamination from the room could have infected the bacteria causing different results. now having a gene for resistance to that antibiotic. and they were finally accepted with the help of heat shock. the sticky ends produced will be complementary and the DNA from the two different sources can be recombined. the cells could grow on the agar plates with ampicillin. there were many lumps in the agar poured into the plates. There were none visible.
Methods: Prepare the agar gel for the electrophoresis by microwaving it for the suggested amount of time. an agarose gel. set the correct voltage and turn on the electricity. but do not let it run all the way out. lambda DNA digested with endonucleases. the whole molecule takes on the negative charge. DNA fingerprinting has occurred. there is placed an agar gel. This gel has wells in it for the samples of DNA to go into. Next. The agarose gel is covered in a buffer so that the DNA is in a neutral pH solution. leaving the DNA stained a royal blue. tracking dye. So. place it in the chamber. Next. when the DNA is placed inside the gel and the electricity turned on so that the poles are drawing the DNA toward the positive side. pour the running buffer over the gel and add the DNA samples into the wells with a micropipette. put the gel into distilled water so that the stain can be taken out of the gel itself. Next. Materials: The materials needed for this lab are the following: an electrophoresis chamber. . Hypothesis: By way of electrophoresis. When the gel has sufficiently hardened. obtain the stain and a staining tray and let the gel set in the stain for a while.enzyme was the first or second etc. Allow this to run until the DNA is almost to the end of the gel. and an electrical supply. Look at and measure the gel over a light box. isolated in that category. and put data into the data table. it will move through the gel and separate according to the size of the fragments. the DNA moves in the direction its charge forces it. micropipette and tips. Since the phosphate groups on the skeleton of DNA are negatively charged. running buffer. In the electrophoresis chamber. the fragments of DNA of lambda can be separated by the traveling of the fragments through agar gel according to fragment size. That way.
269 3.226 5.130 9.614 6.361 2.150 815 580 500 220 Actual base pair sequence 21.207 1.500 11.587 1.000 3.904 1.027 Measured Distance (mm) 12 18 22 28 41 43 Table 6.322 2.530 2.1 HindIII Actual base pairing sequence 23.973 4.2 EcoRI Measured Distance (mm) Band 1 Band 2 Band 3 Band 4 Band 5 Band 6 Band 7 Band 8 12 14 26 28 43 47 49 58 Interpolated base pairs sequence 13.148 or 5.375 .Data: Table 6.557 4.700 3.
Questions: Discuss each of the following factors: Voltage used. Reversal of polarity. and lost to the experiment. the DNA would have been drawn the other way through the gel. If allowed to run longer. If more DNA had been used. If a higher voltage had been used. What could be dome to resolve the fragments? Why would it work? I would take the endonucleases needed to get the two fragment sizes and run an electrophoresis experiment just using those two sizes. Amount of DNA. the bands could merge and be unclear for reading. Two small restriction fragments of nearly the same base-pair size appear as a single band. the bands would have been darker because more of the fragments would have traveled the same distance in the gel. Had the polarity been reversed. and ended up in the running buffer. Running time. the DNA would have eventually ended up into the running buffer. the DNA would have moved faster through the agar gel. It would probably work because these two fragments just by themselves can’t or shouldn’t stay together all the way to the end of the gel. even when the sample is run to the very end of the gel. . and slower if the voltage was low. If not allowed to run long enough. The bands would only have been more distinct and distinguishable.
and the resulting bonds. but some of the few . The gel is helpful because it is like a freeze frame that allows the fingerprinting to be visualized. you could see the change in the band that would be in a different place because it wouldn’t allow the DNA to be cut in that place. A piece of DNA of that size would probably run about 17. 2000. How can a mutation that alters a recognition site be detected by gel electrophoresis? If you ran the normal and the mutant at the same time. What is the source of restriction enzymes? What is their function in nature? They occur naturally in prokaryotes and are used to cut up invading viral DNA that happens to get through the cell wall and plasma membrane of the bacteria.What is a plasmid? How are plasmids used in genetic engineering? Plasmids are small rings of DNA. and 400 base pairs. Show starting point. They are used in genetic engineering because it is considerably easier to manipulate them into taking up preferred genes than it is to change the DNA sequence of the whole cell. positive and negative electrodes.5 millimeters. If a restriction enzyme digest resulted in DNA fragments of the following sizes: 4000. Use the graph prepared from the lab data to predict how far (in mm) a fragment of 8000 base pairs would migrate. This could not be done in liquid or any solid. The electricity is used to pull the DNA in a certain direction so that it will separate. Error Analysis: There were not too many errors that could have occurred in this lab. What are restriction enzymes? How do they work? What are recognition sites? These enzymes are endonucleases that cut the phosphodiether bonds of the DNA. What are the functions of the loading dye in electrophoresis? How can DNA be prepared for visualization? The dye allows the DNA to be more distinct so that accurate measurements can be made in determining the distance traveled and the amount of bands. sketch the resulting separation by electrophoresis. They only cut at specific proteins. Describe the function of electricity and the agarose gel in electrophoresis. 2500. the recognition site.
. other sizes of parts can be hypothesized by following the size of the base pair to the line of best fit drawn on the log sheet. out of order. This tells you about how many millimeters the base pair would probably go if allowed the same circumstances. allowing for a "fingerprint" like uniqueness that is only possible with one’s DNA. Restriction enzymes only cut at their specific protein recognition sites.include the adding DNA to the agar gel. DNA fingerprinting. The wrong DNA samples were added to the wells. Conclusion: In conclusion. or electrophoresis is used to determine the size of the fragments that are cut by restriction enzymes. but the right ones were identified and later labeled correctly. The person transferring had to have a steady hand and good eyes so that the gel wasn’t poked and the DNA made it into the chamber without problems. From the data collected in the electrophoresis experiment. This is useful because no two restriction enzymes code for exactly the same recognition site.
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