Protocol: aTc IV-HSL Responder Cell

Printable Version

Overview

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:

Step	Process	Hands-on Time	Total Time	Notes
0	Prepare glycerol stock	1 hrs	20 hrs
1	Prepare lipids-in-oil solution, outer solution, and substrate stock solutions	2 hrs	2 hrs	Buffers and lipids may be prepared in advance and used for experiments on subsequent days.
2	Assemble PURE reactions	30 min	30 min
3	Encapsulate liposomes	30 min	30 min
4	Measure and image	30 min	6–12 hrs	Total time depends on the exact experiment and incubation conditions. GFP expression should be seen over the first 6 hours at 37C.

Materials and Equipment

DNA	`pT7-tetO-tetO-BjaI`	b. next			[link]
Name	Product	Manufacturer	Part #	Price	Link
Buffers
Glucose	D-(+)-Glucose, 99%	Thermo Scientific	A16828-36	$41.65	[link]
Sucrose	Sucrose, 99%	Thermo Scientific	A15583-36	$41.65	[link]

Lipids
POPC	16:0-18:1 PC (POPC) 25mg/mL	Avanti Lipids	840051C-200mg	$186	[link]
EggPC	Egg PC (95%)	Avanti Lipids	131601C-1g	$391.87	[link]
Liss-Rhod-PE	18:0 Liss Rhod PE 1 mg/mL	Avanti Lipids	810179P-1mg	$273.47	[link]
Mineral Oil	Mineral oil, mixed weight	Thermo Scientific	AC415080010	$53.40	[link]
Glass Syringe 250 µL		Hamilton	14-815-238	$150.15	[link]

PURE
PURE	PURExpress	NEB	E6800S	$295.00	[link]
RNase Inhibitor	RNase Inhibitor, Murine	NEB	M0314S	$81.00	[link]
	`BjaR-GFP-native`	b. next			[link]
OptiPrep	OptiPrep - Density Gradient Media (Iodixanol)	COSMO BIO USA	AXS-1114542	$172	[link]
SAM	S-adenosylmethionine (SAM)	NEB	B9003S	$45	[link]
IV-CoA	Isovaleryl coenzyme A lithium salt hydrate	Millipore Sigma	I9381-10MG	$348	[link]
IV-HSL	3-Methyl-N-[(3S)-tetrahydro-2-oxo-3-furanyl]butanamide	LGC	TRC-M282980-50MG	$171	[link]
DMSO	Dimethyl sulfoxide	Thermo Scientific	042780.M1	$342	[link]
tetR	tetR protein, E. coli (His)	MedChemExpress	HY-P71520A	$100.00	[link]
aTc	anhydrotetracycline	Caymen Chemical	10009542	$44	[link]

Cell culture
XL10-Gold Cells	XL10-Gold µLtracompetent Cells	Agilent	200314	$223	[link]
M9 Media	M9, Minimal Salts, 5X, powder, minimal microbial growth medium	Sigma-Aldrich	M6030-1KG	$260	[link]

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 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:

	Volume to mix (uL)
M9 media	1000
3M Glucose	500

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.

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 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.

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).

Lipids	Stock Concentration (mg/mL)	Volume to add (uL)	Target fraction (%)
Egg PC	25	163.9	70
Cholesterol	50	17.6	29.95
18:0 Liss Rhod PE	1	5	0.05

Heat the lipids-in-oil mixture on a hotplate at 55C / 3 hrs.

Vortex the lipids-in-oil mixture for 1 min.

Prepare outer solution

Final concentration of sugar stock solution is 900 mM

Buffer	Volume to add (uL)
3M Glucose Stock	300
H2O	700

Prepare substrate stock solutions

Substrate	Target Concentration (uM)	MW (g/mol)	Weight (g)	Final Volume (mL)	Note
SAM (32 mM)	5000	398.44	N/A	1
IV-CoA	5000	851.65	4.26	1
IV-HSL	10	183.21	1.83	1
aTc	125	426.4		1	see instructions

SAM is received as a 32 mM stock solution and diluted in ddH2O to a target concentration of 5 mM

IV-CoA is received as a powder 4.26 g dissolved in 1 mL of ddH2O.

IV-HSL is received as a powder 1.83 g and dissolved in 1 mL of DMSO.

aTc is received as a dry powder. Dissolve 57.8 mg aTc in 1 mL of ddH2O to make a 1000x concentrated stock solution. Dilute 10 uL of this solution in 10 mL ddH2O to make a working stock of the appropriate concentration.

Step 3: Assemble PURE Reactions

PURE reaction setup

	Sample (+aTc)	Sample (aTc)	Positive control 1	Positive control 2	Negative control
Component	Volume (uL)	Volume (uL)	Volume (uL)	Volume (uL)	Volume (uL)	Notes
PURE Solution A	12	12	12	12	12	PURE energy solution: small molecules
PURE Solution B	9	9	9	9	9	PURE proteins and ribosomes
RNAse Inhibitor	1.5	1.5	1.5	1.5	1.5	Prevents RNAse activity
pT7-tetO-tetO-BjaI (30 nM)	0.6	0.6	0.6	0	0	tetR-regulated DNA
tetR (30 mM)	4.62	4.62	0	0	0	Repressor
SAM (5 mM)	1.8	1.8	1.8	0	0	Substrate for IV-HSL production.
IV-CoA (5 mM)	0.48	0.48	0.48	0	0	Substrate for IV-HSL production.
OptiPrep	1	1	1	1	1	Adds density for phase-transfer
IV-HSL (10 uM)	0	0	0	0.3	0	Commercial IV-HSL for positive control.
ddH2O	0	0	4.62	7.2	7.5
Total	30	30	30	30	30

Thaw reagents on ice and then keep on ice.

Prepare one microcentrifuge tube on ice for each assembly.

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

Below are the components of the outer solution used in each sample:

aTc (125 uM)	100	0	0	0	0
	Sample (+aTc)	Sample (-aTc)	Positive control 1	Positive control 2	Negative control
	Volume (uL)	Volume (uL)	Volume (uL)	Volume (uL)	Volume (uL)
900mM Glucose	0	1000	1000	1000	1000
M9-Glucose mix	900	0	0	0	0

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

Add 180 µL 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 µL of Outer Solution to each of the tubes labelled O.

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

Centrifuge at 9000 rcf / 4C / 10 min.

Remove the top oil and resuspend the pellet in 100 µL 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 50 µL of M9-cell solution prepared in Step 1 into 384-well glass bottom microplates.

Add 7.5 µL of liposomes made in Step 3 on top of the M9-cell solution in 384-well glass bottom microplates.

Imaging conditions using Operetta:

Temperature: 37 C degree
Green fluorescence channel (200 us, exposure 95%) - excitation: 460 nm - 490 nm; emission: 500 nm - 550 nm.
Red fluorescence channel (50 us, exposure 95%) - excitation: 530 nm - 560 nm; emission: 570 nm - 650 nm.
Brightfield (20 us 95%)
We capture a time lapse over 6 hrs with 10 min intervals.
We also acquired z-stack images spanning from 0 µm to 80 µm of the focal plane.

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). [www.nature.com]

Credits

b.next