Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

Summary

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

Abstract

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

Introduction

Here, we report the procedures for developing a programmable substrate capable of responding to a chemical signal from an engineered cell line.¹ We do this by creating a biotin-streptavidin interface that responds to biotin produced by synthetically engineered Escherichia coli (E. coli) cells. Previously, programmable surfaces have been engineered for a wide range of applications from toxin detection² and point-of-care diagnosis³ to defense and security.⁴ While programmable surfaces can be useful as sensors and actuators, they can be made "smarter" by endow....

Protocol

1. Media and Culture Preparation

1. Prepare lysogeny broth (LB) media by mixing 25 g of LB powder stock with 1 L of deionized (DI) water and autoclaving the solution at 121 °C for 20 min to sterilize.
   1. To prepare LB plates, add 15 g agar (1.5%) to the LB media before sterilization
   2. Prepare stock solutions of 1,000x carbenicillin (Cb) in DI water (50 mg/mL).
   3. If preparing LB media that includes an antibiotic for selection of resistant transformants, wait until the sterilized LB media's temperature is below 60 °C, and then add 1 µL of antibiotic stock for every 1 mL of LB media.
2. For....

Results

Representative results are presented in the accompanying five figures. First, we present the cloning process graphically (Figure 1) so that the reader can visually follow the critical steps for creating the synthetically engineered E. coli strain. In order to characterize the population dynamics of the cells, we provide a growth curve (Figure 2) generated by measuring the optical density at 600 nm (OD₆₀₀) of the population. Then, we sh.......

Discussion

We have presented a new strategy for interfacing engineered living cells with a functionalized material surface. This was accomplished by developing a cell line capable of synthesizing elevated levels of biotin when induced with IPTG. The elevated levels of biotin may then be used to modify the functionalized surface. The protocols detailed how to engineer the E. coli cell line and how to create two different functionalized surfaces.

Critical steps in this protocol occur throughout th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LB Broth, Miller	Fisher Scientific	12-795-027
Agar	Fisher Scientific	BP9744500
Carbenicillin	Fisher Scientific	BP26481
M9, Minimimal Salts, 5x	Sigma-Aldrich	M6030
Casamino Acids	Fisher Scientific	BP1424-100
Magnesium Sulfate, Anhydrous	Fisher Scientific	M65-500
Calcium Chloride, Dihydrate	Fisher Scientific	C79-500
Dextrose (D-Glucose), Anhydrous	Fisher Scientific	D16-1
NEB Turbo Cell Line	New England Biolabs	C2984l
Oligonucleotide Primers	Thermo Fisher Scientific	N/A	25N synthesis, DSL purification
Q5 High-Fidelity Polymerase	New England Biolabs	M0491S
Q5 Reaction Buffer	New England Biolabs	B9027S
dNTP Solution Mix	New England Biolabs	N0447S
Agarose	Bioexpress	E-3120-125
Ethidium Bromide, 1%	Fisher Scientific	BP1302-10
Gel Extraction Kits	Epoch Biolabs	2260250
GenCatch Plasmid DNA Miniprep Kit	Epoch Biolabs	2160250
AatII	New England Biolabs	R0117S
SacII	New England Biolabs	R0157S
HindIII-HF	New England Biolabs	R3104S
EcoRI-HF	New England Biolabs	R3101S
Cutsmart Buffer	New England Biolabs	B7204S
T4 DNA Ligase	New England Biolabs	M0202S
T4 DNA Ligase Reaction Buffer	New England Biolabs	B0202S
ColiRolle Glass Plating Beads	EMD Millipore	7101-3
Glycerol	Fisher Scientific	BP229-1
Isopropyl β-D-1-thiogalactopyranoside (IPTG)	Fisher Scientific	BP1755-10
NHS-Desthiobiotin (DTB)	Thermo Fisher Scientific	16129
Succinimidyl Trans-4-(maleimidylmethyl) Cyclohexane-1-Carboxylate (SMCC)	Thermo Fisher Scientific	S1534
Dimethyl Sulfoxide (DMSO)	Fisher Scientific	BP231-100
Succinimidyl 3-(2-pyridyldithio) Propionate (SPDP)	Thermo Fisher Scientific	S1531
NHS-LC-LC-biotin	Thermo Fisher Scientific	21343
Horseradish Peroxidase (HRP)	Thermo Fisher Scientific	31490
Phosphate Buffered Saline (PBS), 10x Solution	Fisher Scientific	BP399500
Streptavidin (SA)	Thermo Fisher Scientific	21145
Bovine Serum Albumin (BSA)	Fisher Scientific	BP1600-100
Dithiothreitol (DTT)	Fisher Scientific	BP172-5
Ethylenediaminetetaacetic acid (EDTA)	Fisher Scientific	S311-500
Tween 80	Fisher Scientific	T164-500
Hydrogen Peroxide	Fisher Scientific	H325-4
3, 3', 5, 5'-tetramethylbenzidine (TMB)	Fisher Scientific	AC229280050
Vivaspin 500 Centrifugal Concentrators	Viva Products	VS0192
Sodium Acetate, Anhydrous	Fisher Scientific	BP333-500
96-Well Polystyrene Plates	Thermo Fisher Scientific	266120