Controlling localization of Escherichia coli populations using a two‐part synthetic motility circuit: An accelerator and brake

Abstract

Probiotics, whether taken as capsules or consumed in foods, have been regarded as safe for human use by regulatory agencies. Being living cells, they serve as "tunable" factories for the synthesis of a vast array of beneficial molecules. The idea of reprogramming probiotics to act as controllable factories, producing potential therapeutic molecules under user-specified conditions, represents a new and powerful concept in drug synthesis and delivery. Probiotics that serve as drug delivery vehicles pose several challenges, one being targeting (as seen with nanoparticle approaches). Here, we employ synthetic biology to control swimming directionality in a process referred to as "pseudotaxis." Escherichia coli, absent the motility regulator cheZ, swim sporadically, missing the traditional "run" in the run:tumble swimming paradigm. Upon introduction of cheZ in trans and its signal-generated upregulation, engineered bacteria can be "programmed" to swim toward the source of the chemical cue. Here, engineered cells that encounter sufficient levels of the small signal molecule pyocyanin, produce an engineered CheZ and swim with programmed directionality. By incorporating a degradation tag at the C-terminus of CheZ, the cells stop running when they exit spaces containing pyocyanin. That is, the engineered CheZ modified with a C-terminal extension derived from the putative DNA-binding transcriptional regulator YbaQ (RREERAAKKVA) is consumed by the ClpXP protease machine at a rate sufficient to "brake" the cells when pyocyanin levels are too low. Through this process, we demonstrate that over time, these engineered E. coli accumulate in pyocyanin-rich locales. We suggest that such approaches may find utility in engineering probiotics so that their beneficial functions can be focused in areas of principal benefit.

A platform of genetically engineered bacteria as vehicles for localized delivery of therapeutics: Toward applications for Crohn's disease

“…Blots were blocked overnight at 4 °C with Tris‐buffered saline with 0.1% Tween‐20 (TBST) and 10% nonfat milk. Blots against the CheZ protein are described previously …”

“…At the desired time points, a small volume of cells (200 μl) are spun down slowly (400 g) for 5 min, and resuspended in an approximately 40–80 μl volume of chemotaxis buffer (CB; 1× PBS, 0.1 mM EDTA, 0.01 mM l ‐methionine, 10 mM d,l ‐lactate). Videos of fluorescent cells are recorded and analyzed as described previously . For videos of cells using brightfield microscopy, cells were analyzed using a modified version of TumbleScore.…”

“…The majority of such applications solely investigate the production of a biotherapeutic, yet few have addressed targeting mechanisms for localized delivery. We suggest this may be accomplished via a phenomenon known as pseudotaxis, whereby a cell's motility is regulated by a chemical to result in an accumulation of cells within a gradient of the signal; this targeting mechanism has been documented in bacteria with the goals of “smart probiotics” in mind …”

“…That is, we had previously shown that when (a) CheZ deficient cells were restored to swim at speeds near the wild‐type controls (~15 μm/s) due to induced CheZ‐YbaQ production, and (b) accompanying run:tumble ratios were similarly restored to the WT levels, and (c) when swimming was slowed due to CheZ degradation (via inclusion of a degradation tag), pseudotaxis was achieved. These design studies were validated using controlled chemical gradients within a microfluidic device . Accordingly, they enabled subsequent de novo evaluation of alternative genetic circuits based on the engineered cells meeting these established motility metrics.…”

“…As probiotics are designated safe for ingestion and occasionally even administered to patients with IBD, we believe that the methodologies developed here provide robust preclinical designs for uniquely delivering biologics in the GI tract. That is, the experimental platform or “test track” efforts described in this work, in conjunction with our previous design studies, enable engineering assessment of the parameters needed for future animal studies and ultimately, human use.…”