BIO440 Genetics Laboratory Transformation

Transcription

1 BIO440 Genetics Laboratory Transformation The transfer of genetic information between bacteria has been occurring for billions of years. Humans first noticed this process in the laboratory in the 1920 s when Avery and Griffith studied the transformation of avirulent, rough-coated Streptococcus pneumoniae to a smooth-coated and virulent strain. These experiments are now viewed as the first concrete evidence that DNA was the genetic material, but they were not widely recognized until several decades had passed. There are many different ways that DNA can be laterally transferred from one strain to another, including conjugation, transduction, and transformation. In conjugation, the donor has a specific type of plasmid that allows the donor cell to contact the recipient and transfer the DNA directly. In transduction, the donor DNA is packaged into the protein coat of a phage particle, and transferred when that particle infects the recipient. In transformation, 'naked' DNA in the environment is taken up directly by the recipient. All of these processes occur in nature. Interestingly, many bacteria have evolved systems that allow them to regulate their own transformation, and in natural transformations the recipient cell has specific enzymes that mediate the uptake of foreign DNA. These systems vary, but for linear DNA fragments they usually involve the uptake of only one strand of DNA, and the incorporation of the strand into the chromosome via reca -mediated events similar to homologous recombination. While linear DNA can be transformed, plasmid DNA is more stable in different environments, and hence plasmids are frequently the agent of lateral transfer of genetic material. Not all cells in a population will express the genes necessary for transformation to occur; those cells that are in a physiological state allowing them to take up exogenous DNA are said to be competent. In the laboratory, a variety of approaches have been developed for artificial transformation. Artificial transformations can be performed with bacteria that don't undergo natural transformation (such as E. coli) or to increase the frequency of transformation for strains that do. These artificial methods, including electroporation and chemical treatments, damage the cell walls and membranes of recipient bacteria, allowing the transforming DNA to enter the cell. For our transformation of E. coli, we are going to cells that were made competent by concentrating them in an ice-cold solution of calcium chloride. In this procedure, plasmid DNA is added to the calcium chloride/competent cell solution, which is incubated on ice. The divalent calcium cations surround the negatively-charged plasmid DNA molecules, allowing them to associate with the negatively charged bacterial membrane. After allowing the DNA to associate with the membrane for 30 minutes, the cells are heat-shocked by incubating them at 42 C for exactly two minutes. During this heat-shock, transient pores are made in the membrane, allowing the DNA to enter the cells. A five-minute incubation on ice then allows the cells to recover from the heatshock. Cells that have taken up a plasmid can be differentiated from cells that haven't taken up a plasmid by the use of selectable marker genes on the plasmid. The plasmid pgem has a gene that encodes a beta-lactamase that confers ampicillin resistance on the transformed cell. After allowing the cells to grow for 50 minutes in ampicillin-free medium, so that they may express the beta-lactamase gene, the cells are spread onto plates that contain ampicillin, and only transformed cells can form colonies. We are going to plate the transformed cells with the plasmid pgem-plux onto LB + ampicillin plates. You will design and carry out an experiment designed to determine how/if some variable in the protocol affects the number of transformants (cells that took up a plasmid). Each group of

2 two students will do two transformations. One transformation will use 5µl of plasmid containing 5 ng of DNA, and follow the protocol as written below. Your second transformation will have a single variable. Examples of experiments are: same mass of DNA, but in 50 µl volume; omit heat shock; omit 50 min in LB -ampicillin; vortex cells/dna, etc. We will also work with a ligation made using a T/A cloning vector in which PCR products were ligated into pgem. The PCR products were produced by amplifying 16S rdna genes from environmental DNA isolated from Boiling Springs Lake at Lassen Volcanic National Park. Ultimately, we will look at what bacteria/archaea are present in the library of cloned PCR products by sequencing the cloned genes. Our ligation mixture contains a variety of plasmids as well as linear DNA fragments and circular (non-plasmid, non-self-replicating) DNA molecules. The ampicillin marker will enable us to differentiate cells that have taken up a plasmid from nontransformed cells, and from cells that have taken up non-plasmid DNA molecules. Another system allows us to differentiate between the types of plasmids that have been taken up. Because the multiple cloning site (which contains the T/A site that we are trying to clone into) is located in the middle of the beta-galactosidase gene, an insertion into this site will disable, or knock out, the ability of the cell to make beta-galactosidase. The ampicillin-containing plates for this transformation also contain X-gal, which is a synthetic, colorless substrate that forms a blue product when cleaved by beta-galactosidase. We are therefore expecting to see a mix of blue (no insert) and white (+ insert) colonies on our plates. The white colonies are derived from cells that took up an insert-containing plasmid. I will carry out the second transformation and the plates will be available to you. More details are available in the 16S cloning handout. Protocol: Transformation. 1. Obtain three tubes of competent cells from the deep freezer (-85 C). Each tube contains 50 µl of a competent cell solution. Label the tubes with your initials. Also label one tube '+', label one tube '-', and label the final tube 'exp' for. Gently thaw the tubes in your hand, without shaking or mixing. Just as the tubes thaw, place them in ice. 2. Allow the tubes to sit in ice for 3 min. Add 5 µl of your isolated plasmid mix (the mass of which you determined last week) to the '+' tube and the 'exp' tube (unless your experiment involves manipulating that). Mix Gently by stirring for 2 seconds with the end of your pipette tip. Don't add any DNA to the '-' tube. Allow the tubes to incubate on ice for 30 min. 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 five minutes. 5. Add 445 µl of room temperature LB broth to each of the three tubes. Incubate with shaking at 37 C for 50 min. This will allow the cells to express the ampicillin resistance before they are exposed to the ampicillin. 6. Meanwhile get: 7 LB + ampicillin plates, and 2 LB plates. On 6 LB + ampicillin plates, plate: 250 µl, 50 µl, and 10µl of each of your '+' and your 'exp' On the 7th LB + ampicillin plate, plate 250 µl of the '-' transformation On the 2 LB plates, plate: 10 µl of your '+' and your '-' transformation Incubate the plates upside-down at 37 C overnight.

3 Observations and Analysis: Name: What variable did you test? Describe your experiment below. Results LB + Ampicillin plates Vol plated (µl) # colonies standard protocol LB plates # colonies protocol # colonies negative control (10µl) # colonies 10 of standard 10 of Analysis. Transformation efficiency is defined as the number of transformed cells / µg of DNA used in the transformation. Carry out the following calculations to figure out the transformation efficiency of your transformation. Based on colony counts from your 250 µl plates, how many ampicillin resistant cells / ul were present in your transformation tube? Based on this number, what was the total number of transformed cells in your entire 500 µl?

4 Based on colony counts from your 50 µl plates, how many ampicillin resistant cells / ul were present in your transformation tube? Based on this number, what was the total number of transformed cells in your entire 500 µl? Based on colony counts from your 10 µl plates, how many ampicillin resistant cells / ul were present in your transformation tube? Based on this number, what was the total number of transformed cells in your entire 500 µl? Using the highest of the three values determined above, what was the transformation efficiency (#transformants/ug DNA) of the cells used in this experiment? Further questions: 1. What happened when you plated 10 µl of transformed cells onto LB? What does this indicate? 2. What happened in your experiment? Interpret the results.

5 3. Assuming your plasmid was 12,000 bp in size, approximately how many individual plasmid molecules were used in your standard transformation? Your? What fraction of those plasmids was actually taken up in the transformation? (Show calculations) The following questions relate to the 16S rrna gene library & Blue-white screening White colonies Blue colonies LB+ Amp + Xgal/IPTG 1. Does the ratio of white:blue colonies differ from plate to plate? Would you expect it to? Why/why not? What does the ratio of white:blue colonies indicate? Are there some blue colonies that are a paler blue than others? If so, what might this indicate? When the T-tailed pgem vector ligates to itself, does a blue or a white colony result? Why?

6 2. The following scenarios describe various unexpected results that you may have obtained. Try to determine what happened with each of these scenarios and what actions you should take to remedy the problem. a. All of your plates, and all the other students' plates, had no colonies. b. All of your plates had no colonies, but all the other students' plates had the expected results. c. All of your plates, and all the other students' plates, were completely covered with confluent growth -- i.e., there were no distinct colonies. d. All of your plates, and all the other students' plates, had only white colonies. e. All of your plates, and all the other students' plates, had only blue colonies.