BISC 219: Mod 2 Lab 5
The pL4440 Plasmid
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). This particular strain has an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible T7 RNA polymerase gene contained within a stable insertion of a modified lambda prophage λ DE3. 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!
When the E. colicontaining the plasmid are exposed to IPTG they produce large amounts of the T7 RNA polymerase. The T7 RNA polymerase then binds to the plasmid - on either side of our gene of interest and transcribes the DNA into RNA. Because the two single strands of RNA are complementary to each other they will form double stranded RNA.
Background Reading on Bacterial Transformation
During “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.
RNAi Schedule of Experiments
RNAi General Information
Media Recipes
Lab 4: Picking your gene to RNAi
Lab 6: Induction of RNAi plasmid and C. elegans feeding
Lab 7: Single Worm PCR and Agarose Gel Electrophoresis
Lab 8: PCR Reaction Cleanup and Sequencing
