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Prepare a simple flowchart of the experimental procedures for Bacteria Preparation and Transformation

Prepare a simple flowchart of the experimental procedures for Bacteria Preparation and Transformation

Human Anatomy & Physiology (11th Edition)
Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
Chapter1: The Human Body: An Orientation
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Prepare a simple flowchart of the experimental procedures for Bacteria Preparation and Transformation

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The bacterium Echerichia coli (E. coli) is used extensively in molecular biology for recombinant DNA research. E. coli is a normal inhabitant of the human colon and is a common soil bacterium. There are several characteristics of E. coli that makes them extremely useful. The strains used in the lab are non- pathogenic and, hence, pose a minimal risk to lab personnel. They grow at an optimal temperature of 37 °C with no special requirements for nitrogen, oxygen or light. They undergo direct cell division, with one parental cell producing two identical cells and will continue this process given enough nutrients and room for growth. On semi-solid media, an individual bacterium will continue to divide until it forms a colony of bacteria with each member of the colony essentially a clone of the others. E. coli has a single, circular chromosome of approx. 5,000,000 base pairs of DNA, which is less than 0.1 % of the DNA content of a typical human cell. E. coli is easily grown in the lab, either in suspension in a liquid media that provides nutrients such as Luria Broth (LB) or on semi-solid media containing nutrients such as LB agar. E. coli can also contain extrachromosomal, circular, double stranded DNA molecules known as plasmids that typically vary between 1kilobase pairs (kb) to more than 200 kb. Depending on the type of plasmid, they may be present in the bacteria in low copy number or high copy number, and they may replicate autonomously (the majority) or when the bacterium replicates its genomic DNA. Most plasmids have a narrow host range and can only be maintained in a limited number of closely related species. Plasmids are dependent on the enzymes and proteins encoded by their host for their replication and transcription. Over the last 40 years, molecular biologists have learned how to manipulate these plasmids to change the bacteria such that the plasmids carry novel genetic information or foreign DNA that alters the phenotype of the bacteria. The process known as "bacterial transformation" allows one to transmit the recombinant DNA molecules into the bacteria. Transformation can also be used to introduce useful new genes from other species into a species that normally doesn't have those genes. For example, plants and animals can be made resistant to certain diseases by this process. E. coli cannot normally grow in the presence of the antibiotic ampicillin. However, the DNA plasmid PUC19 (see Fig.1) which will be put into the bacteria, contains the gene that encodes ampicillin resistance (ampr). After undergoing transformation, bacteria which successfully take up new DNA will be identified by their ability to grow in the presence of ampicillin. They will form colonies on LB + Ampicillin agar plates that are essentially clones of the original bacterium that was transformed. Even under the best conditions, transformation is inefficient, and plasmids only become established in a minority of the
Transcribed Image Text:The bacterium Echerichia coli (E. coli) is used extensively in molecular biology for recombinant DNA research. E. coli is a normal inhabitant of the human colon and is a common soil bacterium. There are several characteristics of E. coli that makes them extremely useful. The strains used in the lab are non- pathogenic and, hence, pose a minimal risk to lab personnel. They grow at an optimal temperature of 37 °C with no special requirements for nitrogen, oxygen or light. They undergo direct cell division, with one parental cell producing two identical cells and will continue this process given enough nutrients and room for growth. On semi-solid media, an individual bacterium will continue to divide until it forms a colony of bacteria with each member of the colony essentially a clone of the others. E. coli has a single, circular chromosome of approx. 5,000,000 base pairs of DNA, which is less than 0.1 % of the DNA content of a typical human cell. E. coli is easily grown in the lab, either in suspension in a liquid media that provides nutrients such as Luria Broth (LB) or on semi-solid media containing nutrients such as LB agar. E. coli can also contain extrachromosomal, circular, double stranded DNA molecules known as plasmids that typically vary between 1kilobase pairs (kb) to more than 200 kb. Depending on the type of plasmid, they may be present in the bacteria in low copy number or high copy number, and they may replicate autonomously (the majority) or when the bacterium replicates its genomic DNA. Most plasmids have a narrow host range and can only be maintained in a limited number of closely related species. Plasmids are dependent on the enzymes and proteins encoded by their host for their replication and transcription. Over the last 40 years, molecular biologists have learned how to manipulate these plasmids to change the bacteria such that the plasmids carry novel genetic information or foreign DNA that alters the phenotype of the bacteria. The process known as "bacterial transformation" allows one to transmit the recombinant DNA molecules into the bacteria. Transformation can also be used to introduce useful new genes from other species into a species that normally doesn't have those genes. For example, plants and animals can be made resistant to certain diseases by this process. E. coli cannot normally grow in the presence of the antibiotic ampicillin. However, the DNA plasmid PUC19 (see Fig.1) which will be put into the bacteria, contains the gene that encodes ampicillin resistance (ampr). After undergoing transformation, bacteria which successfully take up new DNA will be identified by their ability to grow in the presence of ampicillin. They will form colonies on LB + Ampicillin agar plates that are essentially clones of the original bacterium that was transformed. Even under the best conditions, transformation is inefficient, and plasmids only become established in a minority of the
bacterial population. So, the amp gene acts as a selectable marker that allows these rare transformants to be easily isolated, by killing all the bacteria that were not transformed with the plasmid. In every experiment, it is also essential to include negative controls to eliminate the possibility of contamination. In most cases, "transformation" with the buffer used to resuspend the plasmid DNA is sufficient i.e. no DNA is added to the competent bacteria in the control experiment. No bacteria should grow on the selective plate. Nucleic acids do not enter bacteria such as E. coli without help getting through the outer and inner cell membranes and cell wall. Only once inside the cell can they be expressed and replicate. This process, known as competency, involves a change in the structure and permeability of the cell membrane that allows the plasmid to enter the cell and can be achieved by the treatment with chemicals, such as CaCl2, followed by heat and cold treatments (as first described by Mandel and Higa, 1970), or by physical treatment, such as the use of electric pulses (electroporation). The chemical competency method is simple, rapid and usually generates transformation efficiencies of between 105 and 107 transformed colonies per ug of plasmid DNA. Competent cells can be preserved at -70 °C, although there may be some reduction in their efficiency of transformation during prolonged storage. Your instructor will show you the basics of working with E. coli, sterile technique and plating bacteria. Sterile technique is essential to prevent contaminating bacteria or fungi from growing on your petri plate. Never assume a surface is sterile unless it has been specially prepared (i.e. wiped down with ethanol). Similarly, all glassware should be sterilized by autoclaving or by passing through a flame. Figure 2-1. „Map of the pUC19 plasmid. Glick& Pastemak. lacZ' gene Amp" Multiple cloning sequence gene PUC19 lacI gene Origin of replication
Transcribed Image Text:bacterial population. So, the amp gene acts as a selectable marker that allows these rare transformants to be easily isolated, by killing all the bacteria that were not transformed with the plasmid. In every experiment, it is also essential to include negative controls to eliminate the possibility of contamination. In most cases, "transformation" with the buffer used to resuspend the plasmid DNA is sufficient i.e. no DNA is added to the competent bacteria in the control experiment. No bacteria should grow on the selective plate. Nucleic acids do not enter bacteria such as E. coli without help getting through the outer and inner cell membranes and cell wall. Only once inside the cell can they be expressed and replicate. This process, known as competency, involves a change in the structure and permeability of the cell membrane that allows the plasmid to enter the cell and can be achieved by the treatment with chemicals, such as CaCl2, followed by heat and cold treatments (as first described by Mandel and Higa, 1970), or by physical treatment, such as the use of electric pulses (electroporation). The chemical competency method is simple, rapid and usually generates transformation efficiencies of between 105 and 107 transformed colonies per ug of plasmid DNA. Competent cells can be preserved at -70 °C, although there may be some reduction in their efficiency of transformation during prolonged storage. Your instructor will show you the basics of working with E. coli, sterile technique and plating bacteria. Sterile technique is essential to prevent contaminating bacteria or fungi from growing on your petri plate. Never assume a surface is sterile unless it has been specially prepared (i.e. wiped down with ethanol). Similarly, all glassware should be sterilized by autoclaving or by passing through a flame. Figure 2-1. „Map of the pUC19 plasmid. Glick& Pastemak. lacZ' gene Amp" Multiple cloning sequence gene PUC19 lacI gene Origin of replication
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