Calculating copy number? - (Nov/26/2008 )
I really need some help working out copy number.
I have calculated the number of colonies on my plate, the total number of cells, the weight (g/mol) and concentration (g/ul) of plasmid but I am not sure how to plug these values in to get the number of plasmids per cell?
Copy Number Summary:
1) be sure all the cells are inoculated and grown identically.
2) count the number of cells/mL by microscope rather than determining the number of CFU/mL. Some cells are filamentous and some are non-viable.
3) prepare plasmid DNA by the method in Maniatis' Molecular Cloning rather than using a commercial kit. Incubate Solutions 1, 2 and 3 for 5 minutes each.
4) determine the final DNA concentration by fluorometry rather than A260.
5) convert the DNA concentration from ng/uL to molecules/mL and divide by the number of cells/mL.
Because plasmids grow in bacteria, plasmid copy number is affected by the strain of E. coli used and the presence of advantageous mutations such as endA-, the formulation, osmolarity, and the buffering capacity of the media, the aeration available in the growth chamber, the temperature, the shaking speed, the growth phase at harvest, the age and condition of the inoculum (and whether it is fresh or frozen) and the rate at which concentrations of cells or reagents are changed, nutritional deficiencies, the effects of growth inhibitors (antibiotics) will vary according to how fast the cells are growing, the presence of “poison” genes that inhibit the growth of the bacteria, the presence of strong promoters pointed in the same direction as the plasmid replication transcript, the addition of inhibitors for regulated promoters, the origin of replication, and the skill of the lab personnel in harvesting plasmid DNA. Commercial kits are not designed to harvest 100% of the plasmid DNA in the cell.
Use of an endA- strain of E. coli is preferable when maximum yields are desired, as endonuclease I may contaminate the DNA. Plasmids greater than 10 kb in size are difficult to isolate without shearing some of the plasmid DNA and require special handling.
This means that the nature and volume of the inoculum must be identical. This includes the media lot, the container, aeration or shaking, temperature, etc. If glass containers are used to grow the cells, they should be autoclaved while full of Type I water or rinsed with media prior to use to avoid contamination from residual soap or chemicals in the steam.
Genome copies per cell
The number of E. coli genomes per cell depends on which E. coli strain you are using and what conditions you grow them under. An E. coli chromosome is 4.8 fg. (Vischer et al (1999) J Microscopy 196, pt 1, 61-68, table 1) Stationary phase cells contain 1 genome, exponentially growing cells have more: 1.5 genome equivalents for B/r cells or 2 genome equivalents for other strains such as MC4100. This determination is for cells growing in minimal media. Cells growing in rich media like LB or 2xYT tend to make filaments. E. coli containing very high copy number plasmids also tend to become filamentous. Ultra high copy number plasmids are frequently harvested in a ladder of multimeric forms, indicating a possible titration of topoisomerase. If you have filaments (which will also form if the cells are grown at 42°C), the DNA content can increase to 10 genome equivalents per cell. (Huls et al. (1999) Mol Microbiol 33, 959-970.) Exponentially growing E. coli (in various minimal media) has 1.6 to 4.0 copies, depending on the growth rate. (Churchward et al (1982) J Theor Biol 94, 651-70) To determine the amount of DNA per CFU, you have to know how many genomes are in each bacterium. Likewise, to determine the copy number of a plasmid, you need to decide whether you are concerned with copy number per genome or per cell, whether or not the cells have become filamentous, and how many of the cells are viable.
LB media should not be used for growing ultra high copy number plasmids (2000 copies/cell), as the cells tend to clump and form aggregates in this media. Minimal media, 2xYT, TB, SOB/SOC are all acceptable for these plasmid strains as we have only seen the problem in LB. Cells growing in rich media are larger, have more ribosomes and associated proteins and contain more RNA.
Enumeration of bacteria by microscopic counting is also subject to gross error. Norris and Powell indicate errors arise from reproducibility in filling the chamber and adsorption of bacteria to glass surfaces. Dilutions should be carried out in plastic in high ionic strength medium rather than water, in formaldehyde neutralized with K2HPO4 mixed with a trace of anionic detergent (which also prevents aggregation), or in 0.1 N HCl. Examination by microscopy can be used to determine of the cells are filamentous. Filamentous bacteria are somewhat fragile and the CFU determinations may be artifactually lower after plating. Dead or dying cells contain plasmid DNA but will not form colonies. For detailed descriptions of recommended methods for microscopic counting and the determination of the number of colony forming units (CFU), see A. L. Koch.