Directed evolution of a synthetic phylogeny of programmable Trp repressors

Abstract

As synthetic regulatory programs expand in sophistication, an ever increasing number of biological components with predictable phenotypes is required. Regulators are often 'part mined' from a diverse, but uncharacterized, array of genomic sequences, often leading to idiosyncratic behavior. Here, we generate an entire synthetic phylogeny from the canonical allosteric transcription factor TrpR. Iterative rounds of positive and negative compartmentalized partnered replication (CPR) led to the exponential amplification of variants that responded with high affinity and specificity to halogenated tryptophan analogs and novel operator sites. Fourteen repressor variants were evolved with unique regulatory profiles across five operators and three ligands. The logic of individual repressors can be modularly programmed by creating heterodimeric fusions, resulting in single proteins that display logic functions, such as 'NAND'. Despite the evolutionarily limited regulatory role of TrpR, vast functional spaces exist around this highly conserved protein scaffold and can be harnessed to create synthetic regulatory programs.

Rapid, Low-Cost Detection of Water Contaminants Using RegulatedIn VitroTranscription

“…As an example of this, we were able to use the ROSALIND platform to rapidly validate a putative aTF, CtcS, by showing its ability to bind to an identified operator site and induce with chlortetracycline. In addition, the platform should be easily extensible through further engineering of the aTF or operator sequences to enhance sensitivity, modify specificity, and otherwise tune system performance [36,[72][73][74].…”

“…The above results show that aTFs can be used to regulate IVT in response to ligands through derepression of operator binding. However, there are aTFs, often referred to as aporepressors, that function in the inverse manner-by binding the operator site only when bound to their cognate ligands [36][37][38]. While this ligand corepression represents an important control of gene expression, it is not as useful in a diagnostic biosensor context where direct activation of visible outputs upon exposure to target ligands is often desired.…”

Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes

“…The inherent allosteric mechanism of aTFs can hamper the search of functional mutants with the desired new property from genetic libraries [58,63]. High throughput methods coupled with rounds of negative (for functional DNA binding) and positive (for binding of the new effector) counter-selection have previously been employed to address these challenges by removing non-functional mutants and providing enrichment of desired mutants in the directed evolution of AraC [26,36], PobR [28] and TrpR [29] for example.…”

“…The directed evolution of the aTF to change its substrate specificity is a versatile alternative to develop new biosensors [25][26][27][28][29]. Nonetheless, engineering transcription factors is inherently challenging due to their allosteric nature.…”

“…Small changes in the EBD can affect the stability of the DBD and the performance of the whole system [30]. Examples of aTFs reengineered to accommodate alternative substrates include LacI [31,32], TetR [33,34], AraC [26,35,36] and TrpR [29]. In general, these systems are very well characterised biochemically, structurally, and mechanistically, which greatly facilitates the directed evolution process.…”