• Home
  • Search Results
  • Genetic circuit design automation for the gut resident species Bacteroides thetaiotaomicron.

Genetic circuit design automation for the gut resident species Bacteroides thetaiotaomicron.

Nature biotechnology (2020-04-02)
Mao Taketani, Jianbo Zhang, Shuyi Zhang, Alexander J Triassi, Yu-Ja Huang, Linda G Griffith, Christopher A Voigt
ABSTRACT

Bacteroides thetaiotaomicron is a human-associated bacterium that holds promise for delivery of therapies in the gut microbiome1. Therapeutic bacteria would benefit from the ability to turn on different programs of gene expression in response to conditions inside and outside of the gut; however, the availability of regulatory parts, and methods to combine them, have been limited in B. thetaiotaomicron2-5. We report implementation of Cello circuit design automation software6 for this species. First, we characterize a set of genome-integrated NOT/NOR gates based on single guide RNAs (CRISPR-dCas9) to inform a Bt user constraint file (UCF) for Cello. Then, logic circuits are designed to integrate sensors that respond to bile acid and anhydrotetracycline (aTc), including one created to distinguish between environments associated with bioproduction, the human gut, and after release. This circuit was found to be stable under laboratory conditions for at least 12 days and to function in bacteria associated with a primary colonic epithelial monolayer in an in vitro human gut model system.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
N-Acetyl-L-cysteine, suitable for cell culture, BioReagent
Sigma-Aldrich
Resazurin sodium salt, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Nicotinamide, BioReagent, suitable for cell culture, suitable for insect cell culture
Sigma-Aldrich
Potassium phosphate monobasic, anhydrous, free-flowing, Redi-Dri, ACS reagent, ≥99%
Sigma-Aldrich
Taurocholic acid sodium salt hydrate, ≥95% (HPLC)
Sigma-Aldrich
Menadione, crystalline
Sigma-Aldrich
[Leu15]-Gastrin I human, ≥95% (HPLC)
Sigma-Aldrich
Deoxycholic acid, ≥98% (HPLC)
Sigma-Aldrich
Sodium glycocholate hydrate, ≥95% (TLC)
Sigma-Aldrich
L-Cysteine, BioUltra, ≥98.5% (RT)
Sigma-Aldrich
Lithocholic acid, ≥95%
Sigma-Aldrich
Trypsin solution from porcine pancreas, sterile-filtered, BioReagent, suitable for cell culture, 25 g porcine trypsin per liter in 0.9% sodium chloride
Sigma-Aldrich
Hematin porcine
Sigma-Aldrich
Sodium cholate hydrate, suitable for cell culture, BioReagent
Sigma-Aldrich
Sodium chenodeoxycholate, ≥97%
Sigma-Aldrich
Golgicide A, ≥98% (HPLC)
Sigma-Aldrich
Levobupivacaine hydrochloride, ≥98% (HPLC)