Endy:Measkit draft

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Ideas, Issues, & Questions

• PoPS, polymerase per second, is a derivative unit based on the SI base units of Mole and Second. PoPS represents the number of polymerase molecules that pass any specific position on a nucleic acid per unit time.
  • Q. Is derivative the right word? Compound feels like a better word. See here.

• We are proposing that a relative measurement of promoter activity may be useful for reducing variation (across conditions and settings) in the observed absolute activity of promoters. In practical terms we are proposing that the activity of BBa_J23101 be used as a physical reference standard to which the activity of other promoters may be compared. Instead of measuring the absolute activity of a promoter in PoPS, the activity of the to-be-measured promoter and BBa_J23101 are both measured under the same conditions and a unitless ratio is taken (i.e., PoPS/PoPS []= unitless).
  • Q. What should the resulting, unitless number be called, if anything? Standard Promoter Units, SPUs? Or...
    • This specific question has been tricky to resolve. It may be useful (or dangerous) to make comparisons to other types of measurements, and to identify and discuss the issues which led to their success.

• Example 1. Gel Electrophoresis. Slab gel electrophoresis (PMID 11116182) is now used routinely to separate and estimate the mass or length of proteins, RNA, and DNA. Molecules within samples are separated in space and then observed. Typically, the absolute distance that a molecule travels through a gel is not directly converted to the size of the molecule (with apologies to equipment that allows for direct model-based estimation of length or mass from distance traveled, run time length, or some other observable). Instead, reference standards of known length or mass are used, the distance traveled by both the sample and reference are measured and compared, allowing for a relative measure to be made (thereby accounting for differences in experimental conditions). However, since the length or mass of the reference standard is previously known the relative measure can be converted to an absolute measure of the sample such as base pairs or Daltons.
  • There is an important difference between the activity of a promoter and the length of a fragment of DNA. The distance that a fragment of DNA travels on a gel may vary from one experiment to the next but the underlying length of the DNA molecule being characterized does not change. In contrast, the reported absolute activity of a promoter (as measured in PoPS) may vary greatly across experimental conditions, and such variation may reflect true changes in the underlying activity of the promoter.

• How should we measure promoters under varying measurement conditions?
  • There is likely an analogy here to measuring length using a meter stick with a different (or the same) coefficient of expansion (compared to what, the thing being measured?). An open question is whether J23101 when measured under any conditions should always = 1 SPU (even if the amount of PoPS produced is changing). As I understand this is not the way that it would be done when measuring length - if you measure a meter stick at a temperature where it expands 8X then it is 8 meters long, not 1 meter. However, there seems to still be value in treating J23101 as = 1 SPU across different measurement approaches in that it would allow you to rank order promoters measured in different environments by different researchers. So not sure how to reconcile these two different points.
• How should the reference standard and unit be defined?
  • For instance, we could define the unit of the SPU as the # of PoPS from a reference standard promoter (i.e. J23101) under some standard operating conditions.
  • We could alternatively define the SPU as = 1 PoPS.
  • I think that the first option is likely to be the right one, but I think it could be influenced by the answer to the first question above.