A bacterial dual positive and negative selection system for dCas9 activity.

The engineering of switchable or activatable dCas9 proteins would benefit from a single system for both positive and negative selection of dCas9 activity. Most systems that are used to interrogate dCas9 libraries use a fluorescent protein screen or an antibiotic selection for active dCas9 variants. To avoid some of the limitations of these systems, we have developed a single system capable of selecting for either active or inactive dCas9 variants. E. coli expressing active dCas9 variants are isolated in the positive selection system through growth in the presence of ampicillin. The negative selection can isolate cells lacking dCas9 activity through two separate mechanisms: growth in M9 minimal media or growth in media containing streptomycin. This system is capable of enriching for rare dCas9 variants up to 9,000-fold and possesses potential utility in directed evolution experiments to create switchable dCas9 proteins.

The minimal structure for iodotyrosine deiodinase function is defined by an outlier protein from the thermophilic bacterium Thermotoga neapolitana.

The nitroreductase superfamily of enzymes encompasses many flavin mononucleotide (FMN)-dependent catalysts promoting a wide range of reactions. All share a common core consisting of an FMN-binding domain, and individual subgroups additionally contain one to three sequence extensions radiating from defined positions within this core to support their unique catalytic properties. To identify the minimum structure required for activity in the iodotyrosine deiodinase subgroup of this superfamily, attention was directed to a representative from the thermophilic organism Thermotoga neapolitana (TnIYD). This representative was selected based on its status as an outlier of the subgroup arising from its deficiency in certain standard motifs evident in all homologues from mesophiles. We found that TnIYD lacked a typical N-terminal sequence and one of its two characteristic sequence extensions, neither of which was found to be necessary for activity. We also show that TnIYD efficiently promotes dehalogenation of iodo-, bromo-, and chlorotyrosine, analogous to related deiodinases (IYDs) from humans and other mesophiles. In addition, 2-iodophenol is a weak substrate for TnIYD as it was for all other IYDs characterized to date. Consistent with enzymes from thermophilic organisms, we observed that TnIYD adopts a compact fold and low surface area compared with IYDs from mesophilic organisms. The insights gained from our investigations on TnIYD demonstrate the advantages of focusing on sequences that diverge from conventional standards to uncover the minimum essentials for activity. We conclude that TnIYD now represents a superior starting structure for future efforts to engineer a stable dehalogenase targeting halophenols of environmental concern.

Engineered protein switches for exogenous control of gene expression.

There is an ongoing need in the synthetic biology community for novel ways to regulate gene expression. Protein switches, which sense biological inputs and respond with functional outputs, represent one way to meet this need. Despite the fact that there is already a large pool of transcription factors and signaling proteins available, the pool of existing switches lacks the substrate specificities and activities required for certain applications. Therefore, a large number of techniques have been applied to engineer switches with novel properties. Here we discuss some of these techniques by broadly organizing them into three approaches. We show how novel switches can be created through mutagenesis, domain swapping, or domain insertion. We then briefly discuss their use as biosensors and in complex genetic circuits.