Lab 6: Molecular Biology | Molecular Cloning | Transformation (Genetics)

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Laboratory 6: Molecular Biology
Observing transformations in E. Coli

AP Biology

Spring 2009 INTRODUCTION

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Genome sequencing has been one of the most notable achievements in modern science. Aided with the expanse of faster and faster sequencing machines, researchers have tremendously accelerated the pace of DNA sequencing, which shows much promising research. Advances in DNA technology have made it possible for all of these sequencing achievements to be applied practically. Recombinant DNA, which is DNA whose nucleotides are derived from two distinct sources, often different species, which are combined in vitro into a single DNA molecule, plays an essential role in DNA technology The methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes. In this experiment, plasmid DNA is added to bacteria to observe transformation of naked DNA, which is then incorporated into the bacterial genome. Applications of such genetic engineering allow the manufacture of hundreds of protein products, such as hormones and blood-clotting factors. Using DNA technology, scientists can produce recombinant plasmids containing the genes of interest, and then introduce this into cultured cells which reproduce and replicate the DNA. The expression of the DNA can lead to desirable genes, yielding a desired protein, which can be produced in large quantities and used as needed. Biotechnology is the manipulation of organisms or their components to make useful products to benefit society. The use of microbes to make wine and cheese is perhaps the earliest form of biotechnology that has been applied. Selective breeding of livestock is also another example, which exploits naturally occurring genetic recombination and mutations in species to produce more favorable organisms. Modern biotechnology based on the manipulation of DNA in vitro enables scientists to modify specific genes and move them between organisms as unique as bacteria, plants, and animals. This incredible manipulation of genetic material is the central focus of this lab, which involves the use of Escherichia colito track the intake and integration of DNA into bacterial cells.

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The modern applications of DNA technology have very profound implications on our society. For example, the first genetically engineered medicine was insulin, which has changed the lives of millions of diabetic people over the world. Genetic engineering has also been used to create vaccines, such as one for hepatitis B. Biotechnology also plays significant importance in agriculture, where the creation of genetically modified organisms (GMOs) have allowed for pest resistance and bacterial immunity. Antibiotics are also descended from the work of DNA technology. Criminal law and forensics use DNA technology to solve crimes and analyze mysteries. There are many challenges and problems that a molecular biologist may encounter while studying a particular gene. DNA molecules are incredibly long, and usually carry countless genes. However, genes may occupy only a small proportion of the chromosomal DNA, while the rest may all be non-coding regions. Further complications arise due to the fact that genes are not always completely distinct from surrounding DNA, consisting of very subtle differences in nucleotide sequences. In order to minimize this problem, scientists work directly with single genes by a process called gene cloning. This method prepares well-defined gene-sized pieces of DNA in multiple copies. Bacterial plasmids may only be small, circular DNA molecules that replicated aside from the main bacterial chromosome, but they play a huge role in genetic engineering. A basic overview of gene cloning involves the isolation of a bacterial plasmid from its cell. Foreign DNA is integrated into the plasmid, forming a recombinant DNA molecule, which is then returned to the bacterial cell. This recombinant bacterium reproduces asexually to form a clone of two identical cells. Since the dividing bacteria replicate the plasmid and pass it on to their progeny, the gene is simultaneously being cloned. This process can be useful for two reasons: to produce several copies of a desirable gene and to produce a useful protein product. Isolated copies of this

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cloned gene can then be implanted into an organism, which would then obtain a new metabolic capability. On the other hand, a protein product can be harvested in mass quantities from bacterial cultures possessing the cloned gene which codes for the protein. Gene cloning and genetic engineering was made possible by the discovery of restriction enzymes. Also known as restriction endonucleases, these are enzymes which cut DNA molecules at specific locations. Each restriction enzyme is highly specific, recognizing a particular short DNA sequence, or restriction site, and cutting both DNA strands at specific points on the restriction site. Most restriction sites are symmetrical – the nucleotide sequence is the same on both strands when read in the 5’ →3’. Due to the length of the DNA molecule, it is likely to have several restriction sites, and so will be cut up into several restriction fragments. Sticky ends are formed when restriction enzymes cleave the sugar-phosphate backbone in both DNA strands. These short extensions can form hydrogen bonds with complementary sticky ends, which are then made permanent by DNA ligase. Restriction enzymes and DNA ligase is the key in producing recombinant DNA. The plasmids used in this experiment contain two genes which are useful to bacteria: the ampR gene, which provides resistance against the antibiotic ampicillin, and the lacZ gene, which encodes for the protein β-galactosidase. This enzyme catalyzes hydrolyzes lactose into galactose and glucose, and also X-gal, a synthetic molecular mimic of lactose, into blue products. However, there is a restriction site on the lacZ gene. When the plasmids are removed from the bacteria, a particular restriction enzyme cuts it on the lacZ gene, disrupting the plasmid. The plasmid can then become recombinant or remain recombinant. If it incorporates a restriction fragment, it becomes recombinant, and disrupts the lacZ gene. However, the plasmid doesn’t necessarily have to bond with a restriction fragment. In this situation, the plasmid remains

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nonrecombinant and the lacZ gene remains intact. The plasmids then may or may not be taken back by the bacterial cells.

MATERIALS AND METHODS Two sterile 15-mL tubes were labeled either “+ plasmid” or “- plasmid.” The sterile tube labeled + plasmid was the tube in which plasmid DNA was added at a further stage of the lab. A 1-mL sterile transfer pipette was used to add 250 µL of calcium chloride, which was kept icecold for the time being, into each tube. The purpose of the CaCl2 was to increase cell membrane permeability, inducing competence for DNA uptake, allowing any DNA fragments to enter the cell much quicker than it would unaided. Both tubes were then placed on ice. A starter plate of isolated colonies of E. coli was obtained. Using a sterile plastic inoculating loop, colonies of E. coli were transferred into the + plasmid tube. The cells on the loop were immersed into the calcium chloride solution and vigorously spun in order to dislodge any cell mass and obtain as much bacteria as possible. Caution was taken to avoid the transfer of any agar from the plate along with the cell mass. The cells in the tube were immediately suspended by repeatedly pipetting in and out with another sterile transfer pipette. After the suspension appeared milky white, the tube was examined to ensure that no visible clumps of cells remained in the tube. The + plasmid tube was returned to ice, and the same procedure was followed for the – plasmid tube, creating a cell suspension in both tubes. After both tubes were returned to ice, another sterile inoculating tube was used to add one loopful of plasmid DNA to the + plasmid tube, with a volume of about 10 µL. The plasmid DNA was directly immersed into the cell suspension and mixed with the cells. The + plasmid was returned to ice, and both tubes were incubated on ice for a 15 minute period. While the tubes were incubating, 6 media plates were

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obtained: 2 LB, 2 LB/Amp, and 2 LB/Amp/X-gal. One of each pair was labeled +plasmid, while the other plate of each pair was labeled – plasmid. The LB plates acted as the control, while the others were the experimental plates. Following the 15 minute incubation period, the cells were heat shocked. Both tubes were removed directly from the ice and immersed in a 45˚C water bath for 90 seconds. The tubes were gently agitated while they were in the water bath, and then both were returned back into the ice for 1 or more minutes. Another sterile transfer pipette was used to add 250 µL of Luria broth (LB) to each tube. After the LB was mixed with the suspension, the tubes rested on a test-tube rack for a 10 minute recovery period. Some of the cells were then removed and placed on the plates. Cells from the – plasmid tube were spread on the –plasmid plates, while those in the + plasmid tube were spread on the + plasmid plates. A sterile transfer pipette was used to add about 100 µL of cells from the – plasmid tube each appropriate plate. Another sterile transfer pipette was used to transfer 100 µL of the +plasmid tube to its corresponding plates. Glass beads were sued to spread the bacteria around on the plate by using a back-and-forth shaking motion. After the cell suspension was evenly spread over the agar solution, the plates were allowed to rest for several minutes to allow the cells to become absorbed into the agar. The glass beads were removed and the plates were taped together and incubated over the weekend for the bacteria to grow and divide.

RESULTS The following illustrates my predictions for each plate of bacteria, and the observed result, along with what the ideal observation should have been.

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Prediction: yes
Reason: There is only nutrient broth, so the bacteria will grow regardless. Plasmid DNA is not necessary. Observed Result: Lawn of bacteria

Prediction: yes
Reason: Because a plasmid is unnecessary, bacteria will continue to grow because nothing is restricted it, since it’s just Luria broth. Observed Result: Lawn of bacteria

Prediction: no
Reason: Without the plasmid containing the ampR gene, the bacteria have no protection against the antibiotic and cannot grow. Observed Result: There were several colonies of bacteria, surprisingly

Prediction: yes
Reason: Since they have the plasmid containing the ampR gene, they show resistance to the antibiotic and can grow. Observed Result: There was no bacterial growth, surprisingly

Prediction: no
Reason: Without the plasmid containing the ampR gene, the bacteria have no protection against the antibiotic and cannot grow.

Prediction: yes
Reason: The bacteria will grow because they have the gene for ampicillin resistance. Some colonies may be blue or white, depending on if they are recombinant or nonrecombinant. Observed Result: There was growth, but very few colonies

Observed Result: There was no bacterial growth

NOTE: For plates 3 and 4, my prediction was accurate, yet the observed results were completely opposite of each other, leading me to think that perhaps the two plates were labeled incorrectly or mixed up somehow due to an error in procedure. Otherwise, the rest of the data seems to make sense. Plate 6 had very little colonies, maybe because bacteria were running low and more were used for the other plates. We did not observe any blue colonies, maybe because there weren’t enough bacteria to see blue colonies or that all the plasmids were recombinant. The following expresses the ideal results that should be obtained from this experiment, which will be used for the discussion and the remainder of this report.

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LB - plasmid LB/Amp - plasmid LB/Amp/X-gal - plasmid LB + plasmid LB/Amp + plasmid LB/Amp/X-gal + plasmid

lawn 0 colonies 0 colonies lawn 80 colonies 60 colonies

DISCUSSION Plates Lb – plasmid and Lb + plasmid both have a lawn of bacteria, because neither medium has a threat to bacterial growth. Since they are cultured solely on Luria broth, which is simply nutrient broth, they will continue to grow until lack of resources and waste accumulation diminishes their numbers. Transformation cannot be observed in these two plates, as a plasmid is not necessary for any of these bacteria. The Lb/Amp – plasmid and Lb – plasmid plates have different results because Plate 3 contains the antibiotic ampicillin which will kill bacteria that do not have resistance to this. Since the ampR gene lies on the plasmid, which these bacteria are not given, they have no chance of survival, and therefore 0 colonies were observed. As previously mentioned, Plate 1 has a lawn of bacteria because of the lack of any ampicillin threat. Lb/Amp + plasmid plate allows a chance to observe transformation, which must have occurred if the bacteria are growing in the presence of ampicillin. They have acquired the ampR gene, enabling them to become immune to the antibiotic, and so 80 colonies of bacteria grew compared to 0 colonies where the plasmid wasn’t introduced. There was observable growth in the LB/Amp + plasmid and Lb + plasmid, since there was either resistance to ampicillin or no ampicillin to be resistant to. The LB/Amp/X-gal – plasmid plate had 0 colonies, because the bacteria could not protect against the ampicillin, whereas there was growth in LB – plasmid, since there was no ampicillin threat.

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LB/Amp/X-gal + plasmid supported about 60 colonies, because its bacteria took in the plasmid carrying the gene for ampicillin resistance. However, the LB/Amp/X-gal – plasmid couldn’t support any bacterial colonies because ampicillin would kill the bacteria. There was a slight difference between the LB/Amp/X-gal + plasmid plate and the Lb/Amp + plasmid plate. The former included both white and blue colonies, while the latter only included white colonies. This is because the presence of X-gal allowed bacteria carrying nonrecombinant DNA to hydrolyze it into a blue substance. Bacteria that did have recombinant plasmid DNA would have a disrupted lacZ gene and be unable to do this, and remain white. The same difference applies when Plate 6 is compared with Plate 2, LB + plasmid. The evidence which allows one to identify the bacteria that have taken up the plasmid is any of those bacteria that grow in the presence of ampicillin, because that means they must have acquired the ampR gene from the plasmid through transformation. The phenotype of the transformed colony tells you what type of plasmid it has taken in. If the bacteria are blue in Xgal, it has a nonrecombinant plasmid, with an intact lacZ gene, since it is able to hydrolyze X-gal into a blue product. If the bacteria are white in the presence of X-gal, then it has a dysfunctional lacZ gene, and thus a recombinant plasmid. In order to ensure that transformation has occurred successfully, I would inspect the Lb/Amp + plasmid plate, since those are the ones that require a plasmid in order to grow. If colonies form, transformation has occurred successfully and the bacteria have acquired the plasmid for ampicillin resistance. Transformation efficiency is expressed as the number of antibiotic-resistant colonies per µg of plasmid DNA. The object is to determine the mass of the plasmid that was spread on the experimental plate and that was responsible for the observed colonies. The total mass of plasmid used in this experiment was 0.05 µg. The total volume of the entire cell suspension was 510 µL, which consists of 250 µL of CaCl2, 250 µL of Luria broth, and 10 µL of plasmid DNA. Since

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100 µL of suspension was spread on each plate, the fraction spread is 10/51. The mass of the plasmid in the cell suspension spread is about 0.0098 µg, while the transformation efficiency ranged from 6122% - 8163%. Some factors that might influence transformation efficiency might be the addition of calcium chloride, which induced the bacterial cell to take up the DNA by increasing its membrane permeability. The addition of other chemicals might also impact transformation efficiency. The amount of naked DNA placed into the bacterium’s environment might also play a role. The more DNA, the more efficient the transformation would be.

SUMMARY Transformation in E.coli was observed to see how plasmid DNA can be incorporated into the bacterial genome. This process holds many promising future research in genetic engineering and biotechnology. Manipulation of genes can greatly impact our society, and it is crucial to experiment and research as much as possible in order to successfully use bacteria and other organisms to our advantage. DNA technology is a very contemporary field, and an interesting place to conduct research.

REFERENCES

Campbell, Neil A. 2005. Biology/Neil A. Campbell, Jane B. Reece. 7th ed. San Francisco, CA: Pearson Education, Inc. p. 384-388

Goldberg, Deborah T. 2007. Barron’s AP Biology. 2nd ed. New York, NY: Barron’s Educational Series, Inc. p 415-417

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“Lab Bench Activity: Molecular Biology.” 8 April 2009. http://wps.aw.com/bc_campbell_biology_7/

Pack, Phillip E. 1994. Cliffs Advanced Placement Biology Examination Preparation Guide. Lincoln, Nebraska: Cliffs Notes, Inc. p 447-450

Pack, Phillip E. 2007. Cliffs AP Biology. 3rd ed. Hoboken, NJ: Wiley Publishing, Inc. p 265-266.

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