Part:BBa_K3037006

eGFP

eGFP
Function	Expression, Reporter
Use in	Escherichia coli
RFC standard	Freiburg RFC25 standard
Backbone	pSB1C3
Experimental Backbone	pOCC97
Submitted by	Team: TU_Dresden 2019

Overview

Made by the TU Dresden 2019 adapted to the RFC 25 standard (more information).

Biology

Traditionally isolated from the jellyfish Aequorea victoria, the green fluorescent protein (GFP) has become an important marker for gene expression [1]. However, due to the relatively lower sensitivity of wild-type GFP compared to standard reporter proteins, many different mutants of GFP have been engineered. Thereby, mutations introduced in chromophore of GFP significantly improved its detection and made the protein 35 times brighter than wild-type GFP. The modified fluorescent protein, also known as enhanced GFP (eGFP), appeared to be more sensitive compared to initial detection results [2].

Characterization

Measurement of fluorescence: Performed in the composite BioBrick BBa_K3037005

The purified protein functionality of eGFP in the fusion protein was analyzed in the protein purified using the MBP-tag. First of all, the excitation and emission spectrum were measured with Tecan Plate Reader (Bandwith 20 nm) (Figure 5). The excitation maxima was screened from 400 to 490, being the final excitation maxima 480 nm. The emission maxima was measured from 500 to 600 nm with a final result of 512 nm.

Figure 5: Fluorescense spectrum measurement. Excitation maxima 480 nm. Emission maxima 512 nm.

Sequence

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]

References

[1] Stepanenko OV, Verkhusha VV, Kuznetsova IM, Uversky VN, Turoverov KK (Aug 2008). "Fluorescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular processes". Current Protein & Peptide Science. 9 (4): 338–69

[2] Zhang, G., Gurtu, V., & Kain, S. R. (1996). An Enhanced Green Fluorescent Protein Allows Sensitive Detection of Gene Transfer in Mammalian Cells. Biochemical and Biophysical Research Communications, 227(3), 707–711.