Plasmids are circular pieces of DNA that can replicate in bacteria but are not part of the bacterial chromosome. Plasmids are generally circular molecules with fewer base pairs of DNA than the chromosome and with certain sequence elements (called the origin or ori) that allow the plasmid to replicate within the bacterial cytoplasm. Many naturally occurring plasmids have been modified for the purposes of using them as research tools. For example, a gene encoding resistance to an antibiotic can be added to a plasmid so that bacteria carrying the plasmid will become antibiotic resistant. This modification allows for selection of cells that carry plasmid DNA. A simplified map of the C. elegans RNAi plasmid is below: To enable us to make lots and lots of RNA for RNA interference we need to express our gene at high levels. This is done with a specific strain of E. coli called HT115(DE3). The bacteria cells contain the T7 RNA polymerase gene (contained within a stable insertion of a modified lambda prophage λ DE3) under the control of lac operon regulatory elements. This allows expression of T7 polymerase to be controlled by isopropyl-β-D-thiogalactopyranoside (IPTG), a lactose analogue that induces expression of genes under the control of the lac operon o gene. When IPTG is added, the cells will begin to synthesize lots of T7 RNA polymerase. This T7 RNA polymerase can then bind to the T7 promoter sites on the plasmid and begin to synthesize RNA from both T7 RNA polymerase sites. Because the two single strands of RNA are complementary to each other they will form double stranded RNA within the bacterial cell. Additionally, this particular strain is deficient for the RNAaseIII enzyme that degrades double stranded RNA (dsRNA) in the bacterial cell. This allows for the accumulation of dsRNA in the cell and, thus, our ability to induce and RNAi effect! This E. coli strain carries a tetracyclin resistance gene so these cells can be selected on media containing tetracyclin, while the plasmid contains an ampicillin resistance gene that allows only transfomed cells to grow on media containing ampicillin. Background Reading on Bacterial TransformationDuring “transformation,” a single plasmid enters a single bacterium and, once inside, replicates and expresses the genes it encodes. In this case, the relevant genes expressed are for ampicillin resistance and for the piece of the C. elegans gene of interest. The transformation mixes were given a short time to express these gene products and then were spread on an agar plate that contained nutrients and the antibiotics tetracyclin (encoded by the bacteria) and ampicillin (encoded by the plasmid). Only the cells that incorporated the plasmid DNA and expressed the plasmid genes grew to form colonies of bacteria in the presence of ampicillin. The untransformed bacteria failed to form visible colonies on the ampicillin containing agar surface. Most bacteria do not usually exist in a “transformation ready” state, but the bacteria can be made permeable to the plasmid DNA by exposing them to calcium chloride. Cells that have been treated with calcium chloride or are otherwise capable of transformation are referred to as “competent.” Competent cells are extremely fragile and must be handled gently, i.e. kept cold, not vortexed, etc. The transformation procedure is efficient enough for most lab purposes; with efficiencies as high as 107 transformed cells per microgram of DNA, but it is important to realize that only 1 cell in about 10,000 is successfully transformed. Plasmid Isolation and TransformationLast week, you selected a colony of bacteria that contains a plasmid with a specific C. elegans gene you will RNAi. Now you will isolate the plasmid DNA and transform it into a HT115(DE3)strain for two reasons: we can induce the HT115(DE) strain to make lots of our gene product of interest and because a freshly transformed culture seems to work much better for eventual knockdown of gene expression by dsRNA interference. You may be wondering why we are taking the plasmid out of E. coli and then putting it back into another fresh culture of E. coli. There is a lot scientists don't yet know about the mechanism of dsRNA interference. One thing we do know is that RNAi knockdown works better when you use freshly transformed feeding bacteria that can be manipulated to overexpress plasmid genes of interest, so that's what we'll do. Yesterday you inoculated a few milliliters of LB broth containing bacteria maintaining a genetically engineered plasmid, pL4440, that has been modified to contain an antibiotic resistance gene to ampicillin and all or part of a C. elegans gene that you want to investigate. You added ampicillin to the broth to ensure that the plasmid DNA would be maintained by the transformed cells. Overnight the cells have grown to high density and the plasmid DNA has undergone many replications. However since you started with a single colony of bacteria and that colony grew from a single transformed cell, all the copies of the plasmid DNA in your overnight culture should be identical (“clones” of one another). To isolate the plasmid DNA from an non-inducible bacterial strain, you will perform what is commonly called a “mini-prep”. This term distinguishes the procedure from a “maxi-” or “large scale-prep” which involves a larger volume of cells and additional steps of purification. The overall goal of each “prep” is the same--to separate the plasmid DNA from the chromosomal DNA so that certain genes on the plasmid DNA can be studied further. TO DO TODAY: To isolate plasmid DNA from an overnight culture of cells, the media is removed from the cells by centrifugation. The cells are resuspended in “Solution I” which contains Tris to buffer the cells and EDTA to bind divalent cations in the lipid bilayer, thereby weakening the cell envelope. Upon the addition of “Solution II,” the chromosomal DNA and the plasmid DNA are denatured by the sodium hydroxide, and the cellular proteins and lipids are dissolved by the detergent, sodium dodecyl sulfate (SDS). The pH of the solution is returned to neutral by the potassium acetate in “Solution III.” At neutral pH the SDS precipitates from solution, carrying with it the dissolved proteins and lipids. In addition, the DNA strands renature at neutral pH. The chromosomal DNA, which is much longer than the plasmid DNA, renatures as a tangle that gets trapped in the SDS precipitate. The plasmid DNA renatures normally and stays in a water based solution. In this way the plasmid DNA is separated from the chromosomal DNA, the proteins and the lipids of the cell. Plasmid DNA can be precipitated out of solution in absolute ethanol and then put back into solution (in an appropriate concentration) in water. The ingredients and concentrations of a stock solution of all reagents (such as Solutions I, II, and III) can be found in the Media Recipes section of this section of the wiki.Today you will transform the isolated plasmid DNA, pL4440, into an IPTG inducable strain of E.coli, HT115(DE3), and spread the transformed bacteria onto LB agar media supplemented with both tetracyclin (resistance confered by a gene expressed by the bacteria) and ampicillin (resistance confered by a gene expressed from the plasmid). ProtocolsPart 1: Plasmid DNA Isolation: Only one of your two overnight cultures should appear cloudy with bacterial growth. If your control is cloudy, please inform your instructor.
Part 2: Transformation of isolated plasmid DNA into HT115(DE3): The HT115(DE3) bacterial cells are on the instructor’s bench You will transform some of your plasmid DNA into this strain. The cells are very fragile, so treat them gently. A few weeks ago, the prep staff made a lot of competent HT115(DE3) cells using the Inoue Method Media:Inoue bacterial transformation.doc. Reference: Inoue H., Nojima H., and Okayama H. 1990. High efficiency transformation of Escherichia coli with plasmids. Gene 96: 23-28. The cells were made competent to take up free plasmid DNA by this treatment. This treatment makes the cell wall and membrane more permeable and our transformation efficiency much greater but it weakens the cells; therefore you must keep them cold and mix them gently throughout the transformation (no vortexing!).
What would it mean if you had no colonies on your plate? Normally, you would expect to have around 100 pale color colonies on each plate. If you have at least one well isolated colony on the plate, you’re all set. After the 24 hour growth period the plate should be placed in the rack in the refrigerator labeled with your lab day. You will use a single colony from the plate to make an overnight broth culture on the day before next lab. If you have no colonies on one or more of your plates, please notify your instructor right away. Before leaving lab today, give the rest of your isolated plasmid DNA to your instructor in a labeled microfuge tube. Make sure your tube is labeled with your name, lab day, plasmid name and color coded with a piece of tape in your team color. RNAi Schedule of Experiments |