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<aside> <img src="/icons/light-bulb_gray.svg" alt="/icons/light-bulb_gray.svg" width="40px" /> Overview

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This protocol integrates work described in the Detector Cell (Detector Cells) and Emitter Cell (IV-HSL Emitter Cell). The Emitter Cell reconstituted the BjaI/BjaR quorum sensing components from Bradyrhizobium japonicum to establish IV-HSL-producing synthetic cells (emitters) and IV-HSL-responsive Escherichia coli cells (receivers). The Detector Cell used aTc as an inducer and tetR as a repressor to control expression of a downstream fluorescent reporter.

The Responder Cell contains PURExpress, tetR protein, and the pT7-tetO-tetO-bjaI template. In the presence of aTc, the Responder Cell will produce the BjaI enzyme from the pT7-tetO-tetO-bjaI template, where the train construct is designed to reinforce repression by the tetR protein bound to the tetO operator. Once BjaI is produced, it catalyzes a reaction between the membrane-impermeable IV-CoA and SAM substrates, resulting in the production of membrane-permeable IV-HSL.

E. coli cells expressing BjaR act as receiver cells, providing a simple method to detect IV-HSL production. When BjaR binds to IV-HSL, it induces GFP expression in XL10-Gold cells, resulting in increasing green fluorescence over time.

There are five key stages to making the aTc IV-HSL Responder Cell:

<aside> <img src="/icons/wrench_gray.svg" alt="/icons/wrench_gray.svg" width="40px" /> Materials and Equipment

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<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Step 1: Pre-culture BjaR receiver cells

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• [ ] Prepare glycerol stock of BjaR receiver cells
  • [ ] Transform XL-10 Gold competent E. coli with bjaR-GFP-native:
    • [ ] Add 1 µL – 5 µL containing 1 ng – 100 ng of plasmid DNA bjaR-GFP-native to 50 µL of XL10-Gold cell mixture. Carefully flick the tube 4–5 times to mix cells and DNA. Do not vortex.
    • [ ] Place the mixture on ice for 15 min. Do not mix.
    • [ ] Heat shock at exactly 42C / 40 seconds. Do not mix.
    • [ ] Incubate on ice for 5 minutes. Do not mix.
    • [ ] Pipette 950 µL of room temperature SOC into the cell mixture.
    • [ ] Incubate the cell mixture at 37C / 225 rpm / 60 min.
    • [ ] Warm LB + carbenicillin plates at 37C / 10 min.
    • [ ] Mix the cells thoroughly by flicking the tube and inverting, then perform 3 serial dilutions, ten-fold each (3x 1:10 dilutions) in LB.
    • [ ] Spread 50 µL – 100 µL of each dilution onto an LB + carbenicillin plate and incubate at 37C / ~15 hrs.
    • [ ] Pick a colony from the LB + carbenicillin plate and inoculate a 5 mL culture tube containing the LB with carbenicillin (100 ug / mL).
    • [ ] Incubate the cells at 37 C / 225 rpm / ~15 hrs.
    • [ ] Add 500 μL of the overnight culture to 500 μL of glycerol (50% v/v) in an microcentrifuge tube and gently mix.
    • [ ] Freeze the glycerol stock tube at -80C
• [ ] Prepare M9 Media: 1× M9 salts, 1-thiamine hydrochloride (0.34  mg / mL), casamino acids (0.2%, w/v), MgSO4(2 mM), CaCl2 (100 µM), and glucose (0.4%, w/v) .
• [ ] Prepare the M9-Glucose Mix by balancing osmolarity of the culture media with PURE (inner solution in liposomes) by adding glucose to the M9 media:

• [ ] Use a sterile pipette tip to scrape some of the frozen bacteria off the glycerol stock and inoculate a 1.5 mL microcentrifuge tube containing the M9 media with carbenicillin (100 ug / mL).
• [ ] Gently mix the tube by inverting 5 times. The solution in the tube will be named M9-Cell Solution in the following part.

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Step 2: Prepare lipids-in-oil solution, outer solution, and substrate stock solutions

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Prepare lipids-in-oil (mineral oil) solution

• [ ] Clean glass syringes.
  • [ ] Pour a small amount of EtOH (95%) into a glass container ****(e.g. a 10 mL beaker).
  • [ ] Assemble the glass syringe and prime it by drawing EtOH into the glass syringe, then empty into a waste bottle.