Part:BBa_E0030:Experience

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Applications of BBa_E0030

We fused the promoter PCMV (BBa_K747096) to a YFP reporter (BBa_E0030).

In order to measure its expression, we culture transformed E.coli in agar plates and measured the fluorescense intensity every 45 minutes. After that, we obtained the following data. [Fig.1]

Figure 1 YFP intensity every 45 minutes.

We made three repetitions, so in order to model the equation, we obtained the mean measure, therefore:

Figure 2 Media YFP intensity every 45 minutes.

We analysed the data and using the Wolfram Mathematica software, we obtained the following equation that fits the data. If we plot this equation, we obtain:

Where we can see that the intensity of YFP grows directly proportional to the time and concentration of the molecule.

Figure 3 Intensity YFP vs time.

Characterization by British Columbia iGEM 2012

YFP fluorescence output by British Columbia iGEM 2012

The strong constitutive promoter-EYFP generator (BBa_K804001) includes an enhanced yellow fluorescence protein (BBa_E0030) under the control of a constitutive pTet promoter (BBa_J23118). This composite part's purpose is to constitutively express the YFP(BBa_E0030), which is also available with a strong ribosome binding site (BBa_B0034).

Fluorescence Output of EYFP under a pTet constituitive promoter in co-culture and mono-culture with MetA- auxotroph grown over time : MetA- auxotroph is transformed with the EYFP construct (BBa_K804001) under the constitutive pTet promoter (BBa_J23118). We co-cultured this with TyrA- auxotrophs containing a ECFP construct (BBa_K804000)under the same constitutive pTet promoter; as well as with TrpA- auxotrophs containing a RFP construct (BBa_K081012) under the same constitutive pTet promoter. These cells were grown in minimal media spiked with 10^-3M amino acids (methionine, tryptophan, and tyrosine) . Fluorescence output of EYFP for the three member co-culture, pairwise co-culture, and mono-culture with MetA- auxotrophs are analyzed using a plate reader which measures emission of YFP at a wavelength of 527nm when excited at 514nm.

User Reviews

UNIQ7998db56f2e9e69a-partinfo-00000009-QINU UNIQ7998db56f2e9e69a-partinfo-0000000A-QINU

••••• 2012 iGEM UIUC

The part worked as expected. The way we quantified it through a plate reader with emmission wavelengths. Excitation: 515nm Emission: 528nm We used this part for this project, data collected can be found at http://2012.igem.org/Team:UIUC-Illinois/Results.

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