Overview¶

This protocol reconstitutes 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), implementing the IV-HSL Emitter Cell.

BjaI is expressed inside Emitter Cells containing PURExpress to produce the enzyme BjaI from the template pT7-bjaI. BjaI will catalyze a reaction between the membrane impermeable IV-CoA and SAM substrates to yield membrane permeable IV-HSL.

E. coli cells expressing BjaR act as receiver cells, providing an easy means to detect IV-HSL production. When BjaR binds IV-HSL, expression of a fluorescent reporter gene controlled by a BjaR-regulated promoter is triggered.

Successfully built IV-HSL Emitter Cells will release IV-HSL and induce GFP expression in XL10-Gold cell with increasing green fluorescence over time.

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

Materials and equipment¶

Experimental protocol¶

Step 1: Pre-culture BjaR receiver cells¶

• Prepare glycerol stock of BjaR receiver cells
  • Transform XL-10 Gold competent E. coli with bjaR-GFP-native:
    • Add 1–5 µl containing 1 pg–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 minutes. Do not mix.
    • Heat shock at exactly 42°C for 40 seconds. Do not mix.
    • Place on ice for 5 minutes. Do not mix.
    • Pipette 950 µl of room temperature SOC into the cell mixture.
    • Shake the cell mixture vigorously (250 rpm) at 37°C for 60 minutes.
    • Warm Ampicilin LB agarose plates at 37°C for 10 mins.
    • Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in LB.
    • Spread 50–100 µl of each dilution onto a Ampicilin agarose plate and incubate overnight for ~15 hrs at 37°C.
  • [if we’re including making a glycerol stock, need overnight culture and glycerol stock preparation here]
• Prepare a streak plate from the glycerol stock (reference)
  • Streak a Ampicillin LB plate from the glycerol stock and incubate overnight at 37C.
• Prepare M9 Media containing 1× M9 salts, 0.34 mg/ml−1 thiamine hydrochloride, 0.2% casamino acids, 2 mM MgSO4, 100 µM CaCl2 and 0.4% (wt/vol) glucose.
• Pick a colony from the E. coli streak plate, and inoculate a 5 mL culture tube containing the M9 media with 100 ug/mL carbenicillin.
• Incubate the cells at 37 °C, 225 rpm, for 3 h. Prepare Emitter liposomes while the cells incubate.
• Dilute the culture media with the pre-warmed M9 media until OD600 = ~0.1.
• Balance osmolarity of the culture media with PURE (inner solution in liposomes) by adding glucose to the M9 media:

Step 2: Prepare lipids-in-oil solution, outer solution, and substrate stock solutions¶

Prepare lipids-in-oil (mineral oil) solution

• Clean glass syringes.
  • Pour a small amount of 95% ethanol into a glass container ****(e.g. a 10 mL beaker).
  • Assemble the glass syringe and prime it by drawing ethanol into the glass syringe, then empty into a waste bottle.
• Use glass syringes to add lipids, as shown in the table below, into the 10 ml glass vial containing 1 ml of mineral oil (final lipid concentration is 5 mg/ml).

• Heat the lipids-in-oil mixture on a hotplate at 55 C for 3 hrs.
• Vortex the lipids-in-oil mixture for 1 min.
• The lipids-in-oil mixture can be stored at 4 C for up to 3 days.

Prepare outer solution

Final concentration of sugar stock solution is 900 mM

Prepare substrate stock solutions

Step 3: Assemble PURE Reactions¶

PURE reaction setup

• Thaw reagents on ice and then keep on ice.
• Prepare a PCR strip in a strip holder on ice for assembly of the three reactions (Sample, Negative, Positive).

Step 4: Encapsulate PURE reactions into Liposomes¶

Some tips and tricks can be found in “Hello, world” PURE Liposomes.

• Set up a microfuge tube rack, with three 1.5 mL microfuge tubes per liposome encapsulation:
  • Number the tubes per the number of reactions assembled in Step 3.
  • For each reaction, label the two tubes:
    • I — Oil emulsion
    • O — Outer solution
• Add 30 ul of PURE reactions prepared in Step 3 to tubes labelled I.
• Add 180 uL of the lipids-in-oil mixture on top of the PURE reactions in tubes labelled I and pipette vigorously until the emulsion becomes cloudy.
• Add 300 uL of outer solution to each of the tubes labelled O.
• Add 210 uL of the milky solution carefully on top of the outer solution in the tubes labelled O.
• Centrifuge at 9000 rpm at 4c for 10 mins.
• Remove the top oil and resuspend the pellet in 100 ul of outer solution.
• Collect the liposomes.

Step 5: Measure and Image Liposomes and Cells¶

Imaging using confocal microscopy (Operetta CLS):

While microscopy setups may vary, our performance data was collected using the following configuration.

• Add BjaR receiver cells prepared in Step 1 into 384 Well Glass Bottom Microplates.
• Add 10 uL of liposomes made in Step 3 on top of the receiver cells in 384 Well Glass Bottom Microplates.
• Imaging conditions using Operetta:
  • Temperature: 37 C degree
  • Green fluorescence channel (200 us expsoure 95%) - excitation: 460-490 nm; emission: 500-550 nm.
  • Red fluorescence channel (50 us exposure 95%) - excitation: 530-560 nm; emission: 570-650 nm.
  • Brightfield (20 us 95%)
  • We capture a 6 h time lapse with 10 min intervals.
  • We also acquired z-stack images spanning from 0 µm to 80 µm of the focal plane.

Measuring usinng plate reader (BioTek Cytation 5):

• Add BjaR receiver cells prepared in Step 1 into 96 Well Glass Bottom Microplates.
• Add 10 uL of liposomes made in Step 3 on top of the receiver cells in 96 Well Glass Bottom Microplates.
• Procedures:
  • Temperature: 37 C degree
  • Read the fluorescence intensity from the bottom
  • Excitation wavelength: 485 nm ; Emission wavelength: 528 nm
  • We capture a 6 h time lapse with 5 min intervals

Background protocols¶

• Prepare lipids for use in encapsulation: Lipid Preparation
• Prepare inner and outer buffers: PURE inner and outer solution

Resources and References¶

• Papers
  • Smith, J. M., Hartmann, D. & Booth, M. J. Engineering cellular communication between light-activated synthetic cells and bacteria. Nature Chemical Biology 19, 1138–1146 (2023). pdf

Credits¶

• b.next