Prof. Papoutsakis of the University of Delaware has just disclosed orthogonal multicolor labeling based on FAST, SNAP-tag and HaloTag.  This work consequently provides a major addition to the genetic toolkit of Clostridium.

The biggest limitation in Clostridia study and engineering is the lack of strong, real-time, oxygen-free, fluorescent reporters.  In Appl. Environ. Microbiol. 2019, Papoutsakis had already introduced FAST as a powerful reporting system in C. acetobutylicumC. acetobutylicum is indeed an obligate anaerobe extensively engineered for biofuel production.  Thereafter, in Appl. Environ. Microbiol. 2020, he discloses a full set of three orthogonal multicolor reporters.  Their basic components are FAST, SNAP-tag and HaloTag.  The two latter fluorescent reporters are orthogonal to FAST and to each other, allowing for simultaneous labeling and visualization.  Firstly, Papoutsakis has proven those reporters in C. acetobutylicum and C. ljungdahlii.  Then, the three reporters were successfully combined to measure cell populations in bacterial mixed cultures.

In conclusion, Papoutsakis recalls the challenges demanding orthogonal multicolor labeling:

  1. multi-species tracking within the growing field of microbial co-cultures and microbiomes;
  2. protein localization and tracking in anaerobes; and
  3. identification and development of natural and synthetic promoters, ribosome-binding sites (RBS), and terminators for optimal protein expression in anaerobes.

One can find fluorogens for FAST, e.g., tfLime a.k.a. HMBR, at The Twinkle Factory.