* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download BIO105 Principles of Biology Transformation
Survey
Document related concepts
Therapeutic gene modulation wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Molecular cloning wikipedia , lookup
Microevolution wikipedia , lookup
Genetic engineering wikipedia , lookup
DNA vaccination wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
History of genetic engineering wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Transcript
BIO105 Principles of Biology Transformation Introduction: The transfer of genetic information between bacteria has been occurring for billions of years. Bacterial transformation was observed in 1920’s by Frederick Griffith, who identified the transformation of one strain of avirulent, rough-coated Streptococcus pneumoniae to another strain that was smooth-coated and virulent (=causing disease). The transforming factor was later investigated in depth by Avery and his co-workers in the 1940’s at what is now the Rockefeller University. Extensive experiments and analysis suggested to those investigators that DNA was the heritable material, but their conclusions were not widely recognized or acknowledged Chromosomal DNA from one bacterial strain can be transferred to another, unrelated species of bacterium, but this occurs only infrequently. Linear fragments of DNA (such as chromosomal fragments) are easily broken down outside a living cell, and many agents in the medium surrounding dying cells can modify the structure of DNA. More commonly, plasmid DNA is involved in natural transformations. Plasmids are small circular molecules of DNA that exist in the cell separately from the bacterial chromosome. There can be different kinds of plasmids within the same cell, as well as multiple copies of a single plasmid. Plasmids are often found associated with bacterial cells and are thought to be responsible for considerable diversity and versatility in bacterial metabolism. Plasmids are also used in biotechnology applications to accomplish the rapid transfer of genetic information between bacterial cells. In this lab we are going to carry out a transformation using pLUX. This plasmid is a recombinant plasmid containing the genes from Vibrio fischeri that are involved in bioluminescence. Various Vibrio species are found in marine habitats, both free living and in symbiotic associations. Vibrio fischeri in particular is known to exist in pure cultures within the light emitting organs of squid and several kinds of fish. Bioluminescence has been known for a long time, with an early description of it by Aristotle, who observed that rotten fish glows in the dark! Almost certainly this was an instance of bacterial bioluminescence on the surface of the rotting fish. Biology departments often get calls from members of the general public reporting these same instances of “glowing” flesh on old fish or other materials of marine origin. A variety of organisms have capacities for bioluminescence and many possible explanations of its significance have been advanced. Bioluminescence may be part of an escape response (jellyfish), involved in peculiar pathways of oxidation-reduction (bacteria, dinoflagellates), or as species recognition signals (squid, fish). Anyone who has seen bioluminescence will testify to its fascination! In bacteria, genes for a specific process are often found clustered into an operon. An operon is an arrangement of several genes such that transcription of all the genes can occur from a single promoter; because the cell is able to control production of messenger RNA from all these genes at the same time, this is an effective way to co-regulate the production of many different proteins. The LUX operon of V. fischeri is a set of genes responsible for expression of proteins involved in bioluminescence. Using restriction endonucleases and DNA ligase enzymes, the Vibrio LUX operon has been inserted into the pBR322 plasmid. The pBR322 plasmid also contains a gene for resistance to the antibiotic Ampicillin. In this lab, you will attempt to accomplish the transformation of non-bioluminescent Eschericia coli by getting the E. coli cells to take up the new plasmid (plasmid pLUX). After transforming the E. coli cells, we will spread them onto a nutrient agar plate that contains ampicillin. Ampicillin is a penicillin-like antibiotic that inhibits bacterial growth by interfering with the ability to synthesize new bacterial cell walls. There is a gene on pLUX that encodes a protein which confers ampicillin resistance; because the original E. coli cells are not resistant to ampicillin, only the cells that have taken up pLUX will be able to grow and form colonies on the plate. The ampicillin-resistance gene encodes a protein that is able to cleave ampicillin. It is neceassary to use this 'selectable marker gene' because transformation is a rare event, and only about 1 in every 10 million cells will be transformed using this procedure. It would be very difficult to separate out those few transformed cells from the rest of the cells if we didn't use a selectable marker gene. Cloning DNA in Plasmids. DNA of any origin (including human) can be inserted into a plasmid (a small, selfreplicating circular molecule of DNA) in a process called cloning. The resulting recombinant plasmid is introduced into rapidly reproducing bacterial cells via transformation protocols such as the one described in this exercise. The newly inserted segment will be replicated along with the rest of the plasmid, and proteins encoded by genes on the inserted DNA can be made in the bacterial cells. In our experiment, the genes necessary for the production of luciferase are on the inserted region, and will be expressed by the transformed E. coli, allowing them to glow in the dark. Protocol: Transformation. 1. Obtain two tubes of competent cells from the refrigerator. Each tube contains 200 µl of a competent cell solution. The tubes should be handled with gentleness and tranquility. Label the tubes with your initials. Label one tube 'pLUX’ and the other tube ‘-’. Place the tubes on ice. 2. Allow the tubes to sit in ice for 3 min. Add ~10 µl of pLUX solution to the pLUX tube. This volume contains about 50 ng of plasmid DNA. Allow the tubes to incubate on ice for 30 min. DO NOT shake the tubes or agitate the cells. 3. Transfer the tubes to a 42 °C water bath for EXACTLY 2 min. DO NOT shake the tubes or agitate the cells. 4. Rapidly and gently return the cells to the ice, and incubate for two minutes. 5. Add 400 µl of room temperature LB broth to each of the two tubes. Incubate at 37°C for 50 min. This will allow the cells to express the ampicillin resistance before they are exposed to the ampicillin. 6. Using a new tip, and a freshly sterilized dally rod, use the spread-plate technique to plate 100 µl of your '-' tube (the one that you DIDN't add plasmid to) on one of your LB + ampicillin plates. Label the plate on the bottom (lids can fall off). 7. Using a new tip, and a freshly sterilized dally rod, use the spread-plate technique to plate 100 µl of your pLUX onto an LB plate. Also plate 100 µl of your 'pLUX' tube onto an LB + ampicillin plate. Results: Plate Tube LB +Ampicillin LB pLUX pLUX LB +Ampicillin '-' (negative control) LB + ampicillin transformed cells LB + ampicillin untransformed cells # of Colonies LB transformed cells In the circles above, make a drawing indicating the number and arrangement of your colonies. Indicate which colonies, if any, are bioluminescent. Analysis. 1. What happened when you plated 100 µl of untransformed cells onto LB+ampicillin? What does this indicate? 2. What happened when you plated 100 µl of transformed cells onto LB+ampicillin? How does this compare with your result when you plated 100 µl of untransformed cells onto LB+ampicillin? What does this indicate? 3. What happened when you plated 100 µl of transformed cells onto LB? How does this compare with your result when you plated 100 µl of transformed cells onto LB+ampicillin? What does this indicate? 4. Do the colonies on the LB plate exhibit bioluminescence? Why or why not? Do the colonies on the LB+ampicillin exhibit bioluminescence? Why or why not? 5. What is a bacterial colony? How do these colonies form? 6. Cells in nature undergo transformation. What kinds of advantages might this offer to a population of cells? What are the dangers involved? 7. Many farmers routinely include small amounts of antibiotics in the feed that is given to their animals. Draw a flowchart describing the steps by which such a practice might lead to an increase in the infection of humans by antibiotic-resistant strains of bacteria.