Overview¶

The Emitter Cell produces and releases a chemical signal molecule into the environment. This capacity provides an example of enzymatic small-molecule production, molecule release as a reporter output, inter-cell communication, and co-culture of synthetic cells with living bacteria. The Emitter Cell is largely based on a paper by Jefferson M Smith, Denis Hartmann, and Michael J. Booth: Engineering cellular communication between light-activated synthetic cells and bacteria.

In this first Emitter, the cell produces and releases N-isovaleryl-L-homoserine lactone (IV-HSL). IV-HSL is a branched acyl-homoserine lactone with several advantages for use in the Emitter Cell: it is able to cross the synthetic cell membrane; its uncommon branched-chain structure makes it orthogonal from many other HSLs Lindemann, 2011; and it is able to activate expression in signal receiver cells (in this case, E. coli) at very low (picomolar) concentrations Lindemann, 2011. The IV-HSL signal is received by a population of E. coli cells, which respond by producing a fluorescent output.

The Detector Cells and Emitter together form the basis for an upcoming Responder Cell; a synthetic cell which can detect a molecular input (such as aTc or IV-HSL itself), and produce a molecular output (IV-HSL) in response. Coupling Detector and Emitter modules will enable signal amplification, where a low amount of a molecule of interest can activate a large population of Responder cells and generate an output that is easy to detect. IV-HSL-detecting Responder Cells could also detect the production of IV-HSL from living cells, providing a means to report on their state in co-culture.

Design¶

The Emitter Cell implements the IV-HSL Emitter Module within a synthetic cell. The Emitter Module produces the BjaI enzyme under the control of a constitutive T7 promoter. BjaI produces IV-HSL from two substrate molecules, S-adenosylmethionine (SAM) and isovaleryl coenzyme A (IV-CoA). IV-HSL diffuses out of the cell, through the lipid bilayer.

Usage¶

Protocol¶

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

Step 1: Pre-culture BjaR receiver cells¶

1. 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.

2. Prepare a streak plate from the glycerol stock (reference)

   • Streak a Ampicillin LB plate from the glycerol stock and incubate overnight at 37C.

3. 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.

4. 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.

5. Incubate the cells at 37 °C, 225 rpm, for 3 h. Prepare Emitter liposomes while the cells incubate.

6. Dilute the culture media with the pre-warmed M9 media until OD600 = ~0.1.

7. 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¶

1. 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.

2. Prepare outer solution. Final concentration of sugar stock solution is 900 mM

3. Prepare substrate stock solutions

Step 3: Assemble PURE Reactions¶

PURE reaction setup

1. Thaw reagents on ice and then keep on ice.

2. 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 PURE Cell.

1. 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

2. Add 30 ul of PURE reactions prepared in Step 3 to tubes labelled I.

3. 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.

4. Add 300 uL of outer solution to each of the tubes labelled O.

5. Add 210 uL of the milky solution carefully on top of the outer solution in the tubes labelled O.

6. Centrifuge at 9000 rpm at 4c for 10 mins.

7. Remove the top oil and resuspend the pellet in 100 ul of outer solution.

8. 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.

1. Add BjaR receiver cells prepared in Step 1 into 384 Well Glass Bottom Microplates.

2. Add 10 uL of liposomes made in Step 3 on top of the receiver cells in 384 Well Glass Bottom Microplates.

3. 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):

1. Add BjaR receiver cells prepared in Step 1 into 96 Well Glass Bottom Microplates.

2. Add 10 uL of liposomes made in Step 3 on top of the receiver cells in 96 Well Glass Bottom Microplates.

3. 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

Modules¶

IV-HSL Emitter Module

DNA Components¶

Key Materials¶

Performance Data¶

Emitter Cells were constructed following and co-cultured with E. coli containing the bjaR-GFP-native IV-HSL receiver plasmid. We performed time-series confocal microscopy (Revvity Operetta CLS) over 8 hours, collecting red (Rhodamine-B) and green (GFP) fluorescence, and brightfield images at 40x magnification across multiple fields per well, such that the entirety of each well was imaged. Timepoints were approximately 15 minutes apart.

The Emitter Cell causes E. coli to express GFP in response to IV-HSL.¶

Liposomes exclude E. coli cells from the plate coverslip¶

Background Protocols¶

• Prepare lipids for use in encapsulation: Lipid Preparation

• Prepare inner and outer buffers: PURE inner and outer solution

Credits¶

• Jefferson Smith & Michael Booth (Oxford / UCL)

• b.next