Synthetic cassettes for pH-mediated sensing, counting and containment

Abstract

As pH is fundamental to all biological processes, pH-responsive bacterial genetic circuits enable precise sensing in any environment. Where unintentional release of engineered bacteria poses a concern, coupling pH sensing to expression of a toxin creates an effective bacterial containment system. Here, we present a pH-sensitive kill switch (acidic Termination of Replicating Population; acidTRP), based on the E. coli asr promoter, with a survival ratio of less than 1 in 10 6 . We integrate acidTRP with cryodeath to produce a two-factor containment system with a combined survival ratio of less than 1 in 10 11 whilst maintaining evolutionary stability. We further develop a pulse-counting circuit with single cell readout for each administered stimulus pulse. We use this pulse-counter to record multiple pH changes and combine it with acidTRP to make a two-count acid-sensitive kill switch. These results demonstrate the ability to build complex genetic systems for biological containment.

Controlling the Implementation of Transgenic Microbes: Are We Ready for What Synthetic Biology Has to Offer?

“…Finally, three systems that respond to pH have been reported (Stirling et al, 2020). The first system controls the expression of the toxin Doc with the pH sensitive promoter P asr , achieving a survival ratio of 10 À6 when exposed to pH 5 conditions.…”

“…To counteract the fitness defect imparted from leaked expression of a toxic gene in permissible conditions, an antitoxin should be included in the design of the system. Most natural toxins evolved to function alongside their cognate antitoxin, and this dynamic can be exploited by expressing antitoxin at low levels to negate the effect of a leaked toxin (Gallagher et al, 2015;Stirling et al, 2017Stirling et al, , 2020.…”

“…The individual and combined essentializer, cryodeath, and acidTRP containment systems (Stirling et al, 2017(Stirling et al, , 2020 all use a toxin antitoxin-based approach, resulting in stable growth for over 100 generations. In addition, the construction of these systems explored a method for intelligent design of libraries with promoter variance to achieve the desired balance of expression between toxin and antitoxin.…”

Transcriptional factor engineering in microbes for industrial biotechnology

“…Transcriptomic analysis revealed a substantial proportion of upregulated genes in the nisIPRKFEG cluster and other genes involved in amino acids biosynthesis. 68 Constructing TF-containing genetic circuits for protein sciences The discovery and in-depth elucidation of the lac operon opened up an era for scientists to utilize the genetic circuits that naturally exist in the system to sense different compounds 69 or environmental factors, 70,71 to detect gene deletion 72 and bacterial cell division. 73 Engineered TFs are generally indispensable components in designed genetic circuits.…”

“…The discovery and in‐depth elucidation of the lac operon opened up an era for scientists to utilize the genetic circuits that naturally exist in the system to sense different compounds 69 or environmental factors, 70,71 to detect gene deletion 72 and bacterial cell division 73 . Engineered TFs are generally indispensable components in designed genetic circuits 74,75 …”