Part:BBa_K1094000

Enhanced flavin-binding fluorescent protein (eFbFP)

The reporter eFbFP is a fluorescent protein that uses flavin mono nucleotide (FMN) as a cofacter. The reporter is suitable for use in anaerobic conditions.

The part was cloned into expression vector pJAM1786 (via pDONR207) using the Gateway system by Invitrogen. The plasmid was transformed into E. coli cells. Colony PCR was carried out and colonies were inoculated into liquid culture. The following day the colonies failed to show significantly more fluorescence than three control cultures. Excitation was done with 485 nm and emission peak was measured at 500 nm.

This was unexpected since we previously showed significant fluorescence from a very similar sequence. Due to two internal PstI sites that sequence was not BioBrick compatible. After removing the restriction sites fluorescence was lost even though no change in amino acid sequence was made. Therefore, we expect the lack of fluorescence to be caused cloning/transformation technical issues and not the sequence. Due to time limitation we are not able to support this statement.

Enhanced flavin-binding fluorescent protein is comprehensively described by Mukherje et al. ("Directed evolution of bright mutants of an oxygen-independent flavin-binding fluorescent protein from Pseudomonas putida". Journal of Biological Engineering, 2012, 6:20) and this group should be acknowledged for the development of the reporter.

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
  INCOMPATIBLE WITH RFC[1000]

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