Protocol: IV-HSL Emitter Cell

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Nucleus IV-HSL Emitter Cell Protocol - v0.1.0.pdf

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

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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:

Step	Process	Hands-on Time	Total Time	Notes
1	Pre-culture BjaR receiver cells	30 mins	3.5 hr
2	Prepare lipids-in-oil solution, outer solution, and substrate stock solutions	1 hr	4 h	Buffers and lipids may be prepared in advance and used for experiments on subsequent days.
3	Assemble PURE reactions	30 mins	30 mins
4	Encapsulate liposomes	30 mins	30 mins
5	Measure and image	30 mins	6–12 h	Total time depends on the exact experiment and incubation conditions. GFP expression should be seen over the first 6 hours at 37C.

<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 1–5 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:

	Volume to mix (uL)
M9 media	1000
3M Glucose	293.81

<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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