Microbes and the Tools of Genetic Engineering Quiz

PDF page 518: The science of using living systems to benefit humankind is called biotechnology. Technically speaking, the domestication of plants and animals through farming and breeding practices is a type of biotechnology. However, in a contemporary sense, we associate biotechnology with the direct alteration of an organism’s genetics to achieve desirable traits through the process of genetic engineering. Genetic engineering involves the use of recombinant DNA technology, the process by which a DNA sequence is manipulated in vitro, thus creating recombinant DNA molecules that have new combinations of genetic material. The recombinant DNA is then introduced into a host organism. If the DNA that is introduced comes from a different species, the host organism is now considered to be transgenic. One example of a transgenic microorganism is the bacterial strain that produces human insulin (Figure 12.2). The insulin gene from humans was inserted into a plasmid. This recombinant DNA plasmid was then inserted into bacteria. As a result, these transgenic microbes are able to produce and secrete human insulin. Many prokaryotes are able to acquire foreign DNA and incorporate functional genes into their own genome through “mating” with other cells (conjugation), viral infection (transduction), and taking up DNA from the environment (transformation). Recall that these mechanisms are examples of horizontal gene transfer—the transfer of genetic material between cells of the same generation.

PDF page 518: The science of using living systems to benefit humankind is called biotechnology. Technically speaking, the domestication of plants and animals through farming and breeding practices is a type of biotechnology. However, in a contemporary sense, we associate biotechnology with the direct alteration of an organism’s genetics to achieve desirable traits through the process of genetic engineering. Genetic engineering involves the use of recombinant DNA technology, the process by which a DNA sequence is manipulated in vitro, thus creating recombinant DNA molecules that have new combinations of genetic material. The recombinant DNA is then introduced into a host organism. If the DNA that is introduced comes from a different species, the host organism is now considered to be transgenic. One example of a transgenic microorganism is the bacterial strain that produces human insulin (Figure 12.2). The insulin gene from humans was inserted into a plasmid. This recombinant DNA plasmid was then inserted into bacteria. As a result, these transgenic microbes are able to produce and secrete human insulin. Many prokaryotes are able to acquire foreign DNA and incorporate functional genes into their own genome through “mating” with other cells (conjugation), viral infection (transduction), and taking up DNA from the environment (transformation). Recall that these mechanisms are examples of horizontal gene transfer—the transfer of genetic material between cells of the same generation.

PDF page 518: The science of using living systems to benefit humankind is called biotechnology. Technically speaking, the domestication of plants and animals through farming and breeding practices is a type of biotechnology. However, in a contemporary sense, we associate biotechnology with the direct alteration of an organism’s genetics to achieve desirable traits through the process of genetic engineering. Genetic engineering involves the use of recombinant DNA technology, the process by which a DNA sequence is manipulated in vitro, thus creating recombinant DNA molecules that have new combinations of genetic material. The recombinant DNA is then introduced into a host organism. If the DNA that is introduced comes from a different species, the host organism is now considered to be transgenic. One example of a transgenic microorganism is the bacterial strain that produces human insulin (Figure 12.2). The insulin gene from humans was inserted into a plasmid. This recombinant DNA plasmid was then inserted into bacteria. As a result, these transgenic microbes are able to produce and secrete human insulin. Many prokaryotes are able to acquire foreign DNA and incorporate functional genes into their own genome through “mating” with other cells (conjugation), viral infection (transduction), and taking up DNA from the environment (transformation). Recall that these mechanisms are examples of horizontal gene transfer—the transfer of genetic material between cells of the same generation.

PDF page 519: example, the human insulin gene is inserted into a bacterial plasmid. This recombinant plasmid can then be used to transform bacteria, which gain the ability to produce the insulin protein.

PDF page 518: The science of using living systems to benefit humankind is called biotechnology. Technically speaking, the domestication of plants and animals through farming and breeding practices is a type of biotechnology. However, in a contemporary sense, we associate biotechnology with the direct alteration of an organism’s genetics to achieve desirable traits through the process of genetic engineering. Genetic engineering involves the use of recombinant DNA technology, the process by which a DNA sequence is manipulated in vitro, thus creating recombinant DNA molecules that have new combinations of genetic material. The recombinant DNA is then introduced into a host organism. If the DNA that is introduced comes from a different species, the host organism is now considered to be transgenic. One example of a transgenic microorganism is the bacterial strain that produces human insulin (Figure 12.2). The insulin gene from humans was inserted into a plasmid. This recombinant DNA plasmid was then inserted into bacteria. As a result, these transgenic microbes are able to produce and secrete human insulin. Many prokaryotes are able to acquire foreign DNA and incorporate functional genes into their own genome through “mating” with other cells (conjugation), viral infection (transduction), and taking up DNA from the environment (transformation). Recall that these mechanisms are examples of horizontal gene transfer—the transfer of genetic material between cells of the same generation.

PDF page 519: Herbert Boyer and Stanley Cohen first demonstrated the complete molecular cloning process in 1973 when they successfully cloned genes from the African clawed frog (Xenopus laevis) into a bacterial plasmid that was then introduced into the bacterial host Escherichia coli. Molecular cloning is a set of methods used to construct recombinant DNA and incorporate it into a host organism; it makes use of a number of molecular tools that are derived from microorganisms.

PDF page 519: Restriction Enzymes and Ligases In recombinant DNA technology, DNA molecules are manipulated using naturally occurring enzymes derived mainly from bacteria and viruses. The creation of recombinant DNA molecules is possible due to the use of naturally occurring restriction endonucleases (restriction enzymes), bacterial enzymes produced as a protection mechanism to cut and destroy foreign cytoplasmic DNA that is most commonly a result of bacteriophage infection. Stewart Linn and Werner Arber discovered restriction enzymes in their 1960s studies of how E. coli limits bacteriophage replication on infection. Today, we use restriction enzymes extensively for cutting DNA fragments that can then be spliced into another DNA molecule to form recombinant molecules. Each restriction enzyme cuts DNA at a characteristic recognition site, a specific, usually palindromic, DNA sequence typically between four to six base pairs in length. A palindrome is a sequence of letters that reads the same forward as backward. (The word “level” is an example of a palindrome.) Palindromic DNA sequences contain the same base sequences in the 5ʹ to 3ʹ direction on one strand as in the 5ʹ to 3ʹ direction on the complementary strand. A restriction enzyme recognizes the DNA palindrome and cuts each backbone at identical positions in the palindrome. Some restriction enzymes cut to produce molecules that have complementary overhangs (sticky ends) while others cut without generating such overhangs, instead producing blunt ends (Figure 12.3). Molecules with complementary sticky ends can easily anneal, or form hydrogen bonds between complementary bases, at their sticky ends. The annealing step allows hybridization of the single-stranded overhangs. Hybridization refers to the joining together of two complementary single strands of DNA. Blunt ends can also attach together, but less efficiently than sticky ends due to the lack of complementary overhangs facilitating the process. In either case, ligation by DNA ligase can then rejoin the two sugar-phosphate backbones of the DNA through covalent bonding, making the molecule a continuous double strand. In 1972, Paul Berg, a Stanford biochemist, was the first to produce a recombinant DNA molecule using this technique, combining the SV40 monkey virus with E. coli bacteriophage lambda to create a hybrid.

PDF page 524: • What is the original function of a restriction enzyme?

PDF page 519: Restriction Enzymes and Ligases In recombinant DNA technology, DNA molecules are manipulated using naturally occurring enzymes derived mainly from bacteria and viruses. The creation of recombinant DNA molecules is possible due to the use of naturally occurring restriction endonucleases (restriction enzymes), bacterial enzymes produced as a protection mechanism to cut and destroy foreign cytoplasmic DNA that is most commonly a result of bacteriophage infection. Stewart Linn and Werner Arber discovered restriction enzymes in their 1960s studies of how E. coli limits bacteriophage replication on infection. Today, we use restriction enzymes extensively for cutting DNA fragments that can then be spliced into another DNA molecule to form recombinant molecules. Each restriction enzyme cuts DNA at a characteristic recognition site, a specific, usually palindromic, DNA sequence typically between four to six base pairs in length. A palindrome is a sequence of letters that reads the same forward as backward. (The word “level” is an example of a palindrome.) Palindromic DNA sequences contain the same base sequences in the 5ʹ to 3ʹ direction on one strand as in the 5ʹ to 3ʹ direction on the complementary strand. A restriction enzyme recognizes the DNA palindrome and cuts each backbone at identical positions in the palindrome. Some restriction enzymes cut to produce molecules that have complementary overhangs (sticky ends) while others cut without generating such overhangs, instead producing blunt ends (Figure 12.3). Molecules with complementary sticky ends can easily anneal, or form hydrogen bonds between complementary bases, at their sticky ends. The annealing step allows hybridization of the single-stranded overhangs. Hybridization refers to the joining together of two complementary single strands of DNA. Blunt ends can also attach together, but less efficiently than sticky ends due to the lack of complementary overhangs facilitating the process. In either case, ligation by DNA ligase can then rejoin the two sugar-phosphate backbones of the DNA through covalent bonding, making the molecule a continuous double strand. In 1972, Paul Berg, a Stanford biochemist, was the first to produce a recombinant DNA molecule using this technique, combining the SV40 monkey virus with E. coli bacteriophage lambda to create a hybrid.

PDF page 519: Restriction Enzymes and Ligases In recombinant DNA technology, DNA molecules are manipulated using naturally occurring enzymes derived mainly from bacteria and viruses. The creation of recombinant DNA molecules is possible due to the use of naturally occurring restriction endonucleases (restriction enzymes), bacterial enzymes produced as a protection mechanism to cut and destroy foreign cytoplasmic DNA that is most commonly a result of bacteriophage infection. Stewart Linn and Werner Arber discovered restriction enzymes in their 1960s studies of how E. coli limits bacteriophage replication on infection. Today, we use restriction enzymes extensively for cutting DNA fragments that can then be spliced into another DNA molecule to form recombinant molecules. Each restriction enzyme cuts DNA at a characteristic recognition site, a specific, usually palindromic, DNA sequence typically between four to six base pairs in length. A palindrome is a sequence of letters that reads the same forward as backward. (The word “level” is an example of a palindrome.) Palindromic DNA sequences contain the same base sequences in the 5ʹ to 3ʹ direction on one strand as in the 5ʹ to 3ʹ direction on the complementary strand. A restriction enzyme recognizes the DNA palindrome and cuts each backbone at identical positions in the palindrome. Some restriction enzymes cut to produce molecules that have complementary overhangs (sticky ends) while others cut without generating such overhangs, instead producing blunt ends (Figure 12.3). Molecules with complementary sticky ends can easily anneal, or form hydrogen bonds between complementary bases, at their sticky ends. The annealing step allows hybridization of the single-stranded overhangs. Hybridization refers to the joining together of two complementary single strands of DNA. Blunt ends can also attach together, but less efficiently than sticky ends due to the lack of complementary overhangs facilitating the process. In either case, ligation by DNA ligase can then rejoin the two sugar-phosphate backbones of the DNA through covalent bonding, making the molecule a continuous double strand. In 1972, Paul Berg, a Stanford biochemist, was the first to produce a recombinant DNA molecule using this technique, combining the SV40 monkey virus with E. coli bacteriophage lambda to create a hybrid.

PDF page 519: Restriction Enzymes and Ligases In recombinant DNA technology, DNA molecules are manipulated using naturally occurring enzymes derived mainly from bacteria and viruses. The creation of recombinant DNA molecules is possible due to the use of naturally occurring restriction endonucleases (restriction enzymes), bacterial enzymes produced as a protection mechanism to cut and destroy foreign cytoplasmic DNA that is most commonly a result of bacteriophage infection. Stewart Linn and Werner Arber discovered restriction enzymes in their 1960s studies of how E. coli limits bacteriophage replication on infection. Today, we use restriction enzymes extensively for cutting DNA fragments that can then be spliced into another DNA molecule to form recombinant molecules. Each restriction enzyme cuts DNA at a characteristic recognition site, a specific, usually palindromic, DNA sequence typically between four to six base pairs in length. A palindrome is a sequence of letters that reads the same forward as backward. (The word “level” is an example of a palindrome.) Palindromic DNA sequences contain the same base sequences in the 5ʹ to 3ʹ direction on one strand as in the 5ʹ to 3ʹ direction on the complementary strand. A restriction enzyme recognizes the DNA palindrome and cuts each backbone at identical positions in the palindrome. Some restriction enzymes cut to produce molecules that have complementary overhangs (sticky ends) while others cut without generating such overhangs, instead producing blunt ends (Figure 12.3). Molecules with complementary sticky ends can easily anneal, or form hydrogen bonds between complementary bases, at their sticky ends. The annealing step allows hybridization of the single-stranded overhangs. Hybridization refers to the joining together of two complementary single strands of DNA. Blunt ends can also attach together, but less efficiently than sticky ends due to the lack of complementary overhangs facilitating the process. In either case, ligation by DNA ligase can then rejoin the two sugar-phosphate backbones of the DNA through covalent bonding, making the molecule a continuous double strand. In 1972, Paul Berg, a Stanford biochemist, was the first to produce a recombinant DNA molecule using this technique, combining the SV40 monkey virus with E. coli bacteriophage lambda to create a hybrid.

PDF page 520: Plasmids After restriction digestion, genes of interest are commonly inserted into plasmids, small pieces of typically circular, double-stranded DNA that replicate independently of the bacterial chromosome (see Unique Characteristics of Prokaryotic Cells). In recombinant DNA technology, plasmids are often used as vectors, DNA molecules that carry DNA fragments from one organism to another. Plasmids used as vectors can be genetically engineered by researchers and scientific supply companies to have specialized properties, as illustrated by the commonly used plasmid vector pUC19 (Figure 12.4). Some plasmid vectors contain genes that confer antibiotic resistance; these resistance genes allow researchers to easily find plasmid-containing colonies by plating them on media containing the corresponding antibiotic. The antibiotic kills all host cells that do not harbor the desired plasmid vector, but those that contain the vector are able to survive and grow. Plasmid vectors used for cloning typically have a polylinker site, or multiple cloning site (MCS). A polylinker site is a short sequence containing multiple unique restriction enzyme recognition sites that are used for inserting DNA into the plasmid after restriction digestion of both the DNA and the plasmid. Having these multiple restriction enzyme recognition sites within the polylinker site makes the plasmid vector versatile, so it can be used for many different cloning experiments involving different restriction enzymes. This polylinker site is often found within a reporter gene, another gene sequence artificially engineered into the plasmid that encodes a protein that allows for visualization of DNA insertion. The reporter gene allows a researcher to distinguish host cells that contain recombinant plasmids with cloned DNA fragments from host cells that only contain the non-recombinant plasmid vector. The most common reporter gene used in plasmid vectors is the bacterial lacZ

PDF page 520: Plasmids After restriction digestion, genes of interest are commonly inserted into plasmids, small pieces of typically circular, double-stranded DNA that replicate independently of the bacterial chromosome (see Unique Characteristics of Prokaryotic Cells). In recombinant DNA technology, plasmids are often used as vectors, DNA molecules that carry DNA fragments from one organism to another. Plasmids used as vectors can be genetically engineered by researchers and scientific supply companies to have specialized properties, as illustrated by the commonly used plasmid vector pUC19 (Figure 12.4). Some plasmid vectors contain genes that confer antibiotic resistance; these resistance genes allow researchers to easily find plasmid-containing colonies by plating them on media containing the corresponding antibiotic. The antibiotic kills all host cells that do not harbor the desired plasmid vector, but those that contain the vector are able to survive and grow. Plasmid vectors used for cloning typically have a polylinker site, or multiple cloning site (MCS). A polylinker site is a short sequence containing multiple unique restriction enzyme recognition sites that are used for inserting DNA into the plasmid after restriction digestion of both the DNA and the plasmid. Having these multiple restriction enzyme recognition sites within the polylinker site makes the plasmid vector versatile, so it can be used for many different cloning experiments involving different restriction enzymes. This polylinker site is often found within a reporter gene, another gene sequence artificially engineered into the plasmid that encodes a protein that allows for visualization of DNA insertion. The reporter gene allows a researcher to distinguish host cells that contain recombinant plasmids with cloned DNA fragments from host cells that only contain the non-recombinant plasmid vector. The most common reporter gene used in plasmid vectors is the bacterial lacZ

PDF page 522: white colonies lack a functional beta-galactosidase enzyme, indicating the insertion of foreign DNA within the polylinker site of the plasmid vector, thus disrupting the lacZ gene. Thus, white colonies resulting from this bluewhite screening contain plasmids with an insert and can be further screened to characterize the foreign DNA. To be sure the correct DNA was incorporated into the plasmid, the DNA insert can then be sequenced.