Part:BBa_K3510000

FAST2 reporter gene

This part encodes for the Fluorescence-Activating and Absorption-Shifting Tag (FAST) protein, a 14 kDAsmall protein, that assembles with various fluorogenic ligands (Fluorogens) [1]. Only upon assembly a fluorescent signal is emitted and allows for detection and imaging with high contrast [1].

The parts registry already contains a FAST sequence (BBa_K2992000) provided by team Nottingham (2019). Anyhow, this sequence has been codon optimized for use in genus Clostridium. With many teams working with E. coli, including us, we obtained the sequence of the FAST2 protein from the Twinkle Factory and edited one base pair to eliminate a PstI binding site for compliance to iGEM’s assembly rules. In our project, it was used as a fluorescent tag of the following DNA fragment, either the phytochelatin or chromoprotein in our constructs BBa_KBBa_K3510002-5.

When tagging any protein of interest fluorescently with FAST, the interchangeable fluorogens allow to exchange the spectral properties of the emitted signal, this makes the experimental set-up more flexible [1]. Additionally, the fluorescence signal emitted by FAST does not rely on molecular oxygen, thus enabling fluorescent tagging in anaerobic conditions [1].

Therefore, we understand this tag as an impressive gadget in the synthetic biology toolbox that will be beneficial for future iGEM Teams working in low-oxygen conditions, with anaerobic bacteria, or who want to conduct fluorescence measurements under different ventilation conditions and still obtain comparable results.

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]

References

1. The Twinkle Factory. The science behind FAST and SplitFAST [cited 2020 Oct 19]. Available from: URL: https://www.the-twinkle-factory.com/the-science-behind-fast-and-splitfast.