Competent Cells on Zageno
Competent Cell Applications
Competent cells facilitate processes such as molecular cloning, protein expression, and mutagenesis, all of which require the transfer of nucleic acids amongst cells. This process of transferring DNA or RNA is known as transformation. Exogenous or foreign DNA is transported across the cell membranes (and cell wall if present), usually with extracellular filaments which bind to exogenous DNA and help it cross the DNA membrane. This occurs naturally, and the cell's ability for foreign DNA uptake is known as its competency. The efficiency of these so-called competent cells is measured as the number of colonies formed per μg of supercoiled DNA. These cells are often used to amplfy specific PCR products of interest.
Thus, competent cells are typically E. coli cells with high competency values. Such values though are normally achieved artificially in the lab, via chemical or electrical means.
Chemically competent cells are prepared via a solution containing CaCl₂ and other salts around 0 ⁰C. The Ca 2+ ions in solution help create pores within the cell membrane, allowing more entryways for foreign DNA to be taken up.
Electrocompetent cells are prepared via electroporation. This requires a specific device which sends a pulse of electricity across the cells, which in theory disrupt the membrane, allowing the foreign DNA to pass through.
Like most things, one procedure is not necessarily better than the other; rather it mostly depends on your laboratory setup. Preparing electrocompetent cells is usually more efficient, yet require expensive equipment. Preparing chemically competent cells is cheaper and does not require special equipment, yet is less efficient.
Competent Cell Types
The E. coli strains which are optimized for cloning typically have recA and endA markers, which increase plasmid DNA quality and stability. Furthermore, competent cells optimized for cloning often have screening techniques implemented into the exogenous DNA. The most common being blue / white screening.
Blue-white screening is a common technique to detect whether your cells have absorbed foreign DNA. DNA of interest containing a special sequence is ligated into a vector, which is then absorbed by your competent cells. These are then grown with X-gal. Those successfully transformed will produce white colonies; cells containing no exogenous DNA will appear blue.
E. coli strains optimized for cloning are not well suited for protein expression, so when looking for the right strain for your experiment, be sure to choose the one most appropriate. Competent cells for protein expression are typically engineered to withstand high protein levels, and can also have greater transcriptional control and protein folding.
For anyone just beginning their competent cell experiments, genotype-nomenclature can seem extremely daunting, which is understandable when:
F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araΔ139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG tonA::Ptrc-ccdA
is the genotype. So, the following is a quick guide to help you start decoding the meaning behind the symbols used in naming genotypes.
Genes: designated by three letters in lower case (usually related to the phenotype or pathway). Different genes involved in the same pathway are separated via a capital letter after the assigned three letter name.
Ex: Mutations involving the synthesis of pyrimidine are so named pyr. One gene involved with the productions of an enzyme is named pyrC while another gene encoding for a different enzyme is pyrD.
Alleles: Every allele mutation is assigned a number. In order to prevent confusion and error, for every pyr mutation, a unique number is assigned and not reused.
Phenotype: when denoting the phenotype of a particular gene, the abbreviation is not italicized and has the first letter capitalized (DnaA – protein produced by the dnaA gene)