Reporter

Part:BBa_E0430

Designed by: Caitlin Conboy   Group: Antiquity   (2004-03-15)


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:

ratiometric construct
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.

CFP better than 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


Team: Tianjin 2019 characterization

Team:Tianjin 2019 contributed to the reporter's characterization by connecting it to the combined promoters J23110 and J23115, assembling and testing their fluorescence intensity and detailed fluorescence excitation curves.

Figure 1. Characterization scheme used by Team Tianjin

We linked the constructed fragment to the plasmid pRS415 and transformed it into Match 1 strain. Under normal culture conditions (LB+amp solid medium, 37℃), the Match 1 strain was cultured overnight. After about 8 hours, the colony was visible to the naked eye, with no obvious difference in appearance from ordinary e. coli (EYFP was not visible to the naked eye).

The experiment design
1.PCR validation
Because EYFP was not visible with the naked eye, we performed colony PCR validation to confirm whether the transformation was successful.

Fig2. PCR validation results of the corresponding gene (strain 22,46,54 has the corresponding gene length band)

2.Determination by fluorescence microplate reader
The bacteria verified in the previous step were cultured separately overnight, then a small number of bacteria were selected and suspended in 200μL ddH2O, and the fluorescence intensity of the same bacteria was measured under emission/excitation of 527/514nm (reference value).

Fig 3. Fluorescence intensity comparison of EYFP under the control of two constituent promoters

3. Fluorescence curve measurement
Under the condition of fixed excitation wavelength (512nm), we continuously measured its specific fluorescence intensity at the emission wavelength of 470~600nm, and obtained the following results.

Fig4. Fluorescence curve of EYFP expression
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Categories
//classic/reporter/ret
Parameters
biology
emissionYellow
tagNone
typeReporter