Part:BBa_E0430

EYFP (RBS+ LVA- TERM) (B0034.E0030.B0015)

Standard YFP Output Device -LVA tag

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Team USP-Brazil 2018 Improvement

Team USP-Brazil, in 2018, has improved this part (BBa_K2771020) by adding it to a control gene, being that CFP constitutively expressed by ptet, which serves as a proxy for external influences on the first reporter's measurement, such as cell density, plasmid copy number and ,to some amount, metabolic burden:

Figure 1: Scheme of USP-Brazil's improvement BBa_K2771020

This improvement has shown to give better measurements when normalizing YFP output by dividing by CFP fluorescence than when just measuring YFP and dividing that value by the measured OD600. This gives us the possibility of a much more reproducible result, less dependant on growth media and cell density (a rather unreliable parameter to measure) at the time of each measurement. Another good point for using this is that dividing fluorescence measurements gives us an adimensional parameter, that has greater capacity for comparation with other experiments and constructs. In our measurements of quorum-sensing-responsive promoters activity, we compared the variance of our controls, which should have a constant value for fluorescence due to a constant amount of leakiness, when normalizing by the OD600 value and the CFP fluorescence measurement. We found, in this and other experiments, that the normalization using CFP presented significatively less variance. In this example, we found a variance that represented, when reaching equilibrium value, a percentage of the mean twice as small as with the normalization with the OD.

Figure 1: Graphs show activity of an expression pattern that should be constitutive. When plateauing, it is clear that the normalization by CFP gives a steadier control and a better proxy for cell conditions than OD