Propagation of Recombinant Plasmids

Found in some bacteria and yeast species, plasmids are comparatively small, self-replicating rings of DNA located outside a cell's main chromosome. They are highly useful in genetic engineering because it is relatively easy to splice selected genes from other species into plasmids, transfer them among host cells from the same or different species, and stimulate them to replicate--or propagate--at much higher-than-normal--or amplified--rates. Recombinant plasmids are simply those that have been engineered to contain genes from other species. Especially with amplified propagation, recombinant plasmids can more rapidly and readily express desired proteins on a large, industrial scale.

Things You'll Need

• Preexisting plasmid and gene libraries, or plasmids and genes you have isolated yourself from their host organisms and spliced together
• Cell culture equipment
• Amplification agent(s) such as calcium chloride, electrical shock, cold and heat, chloramphenicol, electrical shock, and/or Taq polymerase enzyme

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Instructions

• 1

  Choose the amplification procedure or procedures that best fit your specific recombinant plasmid and host organism. Some procedures work better with certain plasmids and hosts than with others. If you are working with a novel, still-experimental recombinant plasmid and/or are trying out a different host species, you may have to determine the best amplification process or processes through trial and error.

• 2

  Try the process called transformation if your specific plasmid and host cell species might respond to it. Chill the cells and plasmids to zero degrees C in a calcium chloride (CaCl) solution. Quickly raise the temperature to 37-43 degrees C. This abrupt temperature change causes the cell membranes to take up larger quantities of the plasmid.

• 3

  Send high-voltage electricity pulses through the solution of plasmids and cells if electroporation is the desired amplification method. This technique increases cell membrane permeability to the plasmids.

• 4

  Expose the host cell culture to low levels of the antibiotic chloramphenicol to generate as many as one hundred plasmids per cell. This method suppresses replication of the host cells' main chromosomes while inducing uncontrolled replication of the plasmid, including the gene spliced into it.

• 5

  Apply the polymerase chain reaction or PCR method to generate plasmid copies at very high orders of magnitude. PCR is based on DNA polymerization, the chemical reaction through which genetic sequences form in nature. PCR is most often catalyzed by a heat-stable enzyme called the Taq polymerase, extracted from a bacterial species that lives in hot springs.