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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The protocol describes the formation of robust and biocompatible DNA-laden microcapsules as multiplexed in vitro biosensors capable of tracking several ligands.

Abstract

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical cores. Following adsorption of a prime layer and DNA plasmids, the formation of robust microcapsules was facilitated by inducing β-sheets in silk secondary structure during acute dehydration of a single silk layer. Hence, the layering occurred via multiple hydrogen bonding and hydrophobic interactions. Upon adsorption of multilayered shells, the core-shell structures can be further functionalized with gold nanoparticles (AuNPs) and/or antibodies (IgG) to be used for remote sensing and/or targeted delivery. Adjusting several key parameters during sequential deposition of key macromolecules on silica cores such as the presence of a polymer primer, the concentration of DNA and silk protein, as well as a number of adsorbed layers resulted in biocompatible, DNA-laden microcapsules with variable permeability and DNA loadings. Upon dissolution of silica cores, the protocol demonstrated the formation of hollow and robust microcapsules with DNA plasmids immobilized to the inner surface of the capsule membrane. Creating a selectively permeable biocompatible membrane between the DNA plasmids and the external environment preserved the DNA during long-term storage and played an important role in the improved output response from spatially confined plasmids. The activity of DNA templates and their accessibility were tested during in vitro transcription and translation reactions (cell-free systems). DNA plasmids encoding RNA light-up aptamers and riboswitches were successfully activated with corresponding analytes, as was visualized during localization of fluorescently labeled RNA transcripts or GFPa1 protein in the shell membranes.

Introduction

The field of synthetic biology offers unique opportunities to develop sensing capabilities by exploiting natural mechanisms evolved by microorganisms to monitor their environment and potential threats. Importantly, these sensing mechanisms are typically linked to a response that protects these microorganisms from harmful exposure, regulating gene expression to mitigate negative effects or prevent intake of toxic materials. There have been significant efforts to engineer these microorganisms to create whole-cell sensors taking advantage of these natural responses but re-directing them to recognize novel targets and/or to produce a measurable signal that can be measured....

Protocol

1. Construction of plasmid vector.

  1. Construct a plasmid vector (pSALv-RS-GFPa1, 3.4 kb) by amplification of the coding sequence of a theophylline riboswitch (ThyRS) coupled with GFPa1 from pJ201:23976-RS-GFPa1 vector (designed and created by DNA2.0) and insertion into E. coli expression vector, pSAL13. Use forward (5'-CGTGGTACCGGTGATACCAGCATCGTCTTGATG-3') and reverse (5'-CGTGCTCAGCTTAAGCCAGCTCGTAG-3') primers to amplify the coding sequence of ThyRS coupled with .......

Representative Results

Here, the study addresses the functionality of DNA templates encoding different sensor designs (two types of RNA-regulated transcription/translation elements) after encapsulation in silk protein capsules. Microcapsules were prepared via templated Layer-by-Layer (LbL) assembly of the key components: A prime layer, DNA plasmids encoding sensor designs, and silk fibroin biopolymer (Figure 2). Deposition of macromolecules in a layered fashion allows controlling the permeability of the capsule me.......

Discussion

Selectively permeable hydrogel microcapsules loaded with various types of DNA-encoded sensor designs can be prepared following this protocol. One of the distinctive features of the LbL approach is the ability to tailor the complexity of microcapsules during the bottom-up assembly, which usually starts with the adsorption of molecular species on sacrificial templates. By carefully adjusting concentrations of the initial components, pH conditions, and the number of layers, microcapsules with different DNA loading parameter.......

Acknowledgements

This work was supported by LRIR 16RH3003J grant from the Air Force Office of Scientific Research, as well as the Synthetic Biology for Military Environments Applied Research for the Advancement of S&T Priorities (ARAP) program of the U.S. Office of the Under Secretary of Defense for Research and Engineering.

The plasmid vector sequence for ThyRS (pSALv-RS-GFPa1, 3.4 kb) was generously provided by Dr. J. Gallivan. Silkworm cocoons from Bombyx mori were generously donated by Dr. D.L. Kaplan from Tufts University, MA.

....

Materials

NameCompanyCatalog NumberComments
(Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4 H)-one (DFHBI-1T)LucernaDFHBI-1T
5x T4 DNA Ligase BufferThermoFisher Scientific46300-018
6x Blue Gel Loading DyeNew England BioLabsB7021S
96-well plates, black circularCorning3601
AgaroseSigma-AldrichA9539BioReagent, for molecular biology, low EEO
Ampicillin sodium saltSigma-AldrichA0166powder or crystals, BioReagent, suitable for cell culture
BlpI restriction enzymesNew England BioLabsR0585S
Corning Disposable Vacuum Filter/Storage SystemsFisherScientific09-761-1
Dimethyl sulfoxide, DMSOSigma-Aldrich472301ACS reagent, ≥99.9%
DNA Plasmid, pET28c-F30-2x Broccoli (5.4 kb), BrocApt.AddgenePlasmid #66788
DyLightTM550 Antibody Labeling kit (Invitrogen)ThermoFisher Scientific84530
E. coli S30 extract system for circular DNAPromegaL1020
Falcon Conical centrifuge tubes, 15 mLFisherScientific14-959-53A
Falcon Conical centrifuge tubes, 50 mL14-432-22
Fisherbrand Microcentrifuge tubes, 1.5 mLFisherScientific05-408-129
Hydrofluoric acid, HFSigma-Aldrich695068ACS reagent, 48%
Kanamycin sulfateSigma-Aldrich60615mixture of Kanamycin A (main component) and Kanamycin B and C
KpnI restriction enzymesNew England BioLabsR0142S
LB agar plate supplemented with 100 µg/mL ampicillinSigma-AldrichL5667pre-poured agar plates with 100 µg/mL ampicillin
LB agar plate supplemented with 50 µg/mL kanamycinSigma-AldrichL0543pre-poured agar plates with 50 µg/mL kanamycin
LB broth (Lennox grade)Sigma-AldrichL3022
Lithium bromide, LiBrSigma-Aldrich213225ReagentPlus, ≥99%
Max Efficiency DH5-α competent E. coli strainThermoFisher Scientific18258012
MethanolMilliporeSigma322415anhydrous, 99.8%
MilliQ-waterEMD MilliPoreMilli-Q Reference Water Purification System
MinElute PCR Purification KitQiagen28004
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, EDCSigma-AldrichE1769
PBS (phosphate buffered saline)ThermoFisher Scientific100100231x PBS, pH 7.4
Phusion High-Fidelity DNA PolymeraseNew England BiolabsM0530S
Polyethylenimine, branchedSigma-Aldrich408727average Mw ~25,000
PURExpress In Vitro Protein Synthesis KitNew England BioLabsE6800S
QIAEX II Gel Extraction KitQiagen20021
QIAprep Spin Miniprep KitQiagen 27104
Quick-Load 2-Log DNA Ladder (0.1-10.0 kb)New England BioLabsN0469S
SiO₂ silica microspheres, 4.0 µmPolysciences, Inc.24331-1510% aqueous solution
Slide-A-Lyzer G2 Dialysis Cassettes, 3.5K MWCO, 15 mLThermoFisher Scientific87724
Sodium carbonate, Na₂CO₃Sigma-Aldrich222321ACS reagent, anhydrous, ≥99.5%, powder
Spectrum Spectra/Por Float-A-Lyzer G2 Dialysis DevicesFisherScientific08-607-008Spectrum G235058
SYBR Safe DNA gel stainThermoFisher ScientificS33102
T4 DNA Ligase (5 U/µL)ThermoFisher ScientificEL0011
TheophyllineSigma-AldrichT1633anhydrous, ≥99%, powder
Tris Acetate-EDTA buffer (TAE buffer)Sigma-AldrichT6025Contains 40 mM Tris-acetate and 1 mM EDTA, pH 8.3.
UltraPure DNase/RNase-Free Distilled WaterFisherScientific10-977-023
ZymoPURE II Plasmid MaxiPrep kitZymoResearchD4202

References

  1. Slomovic, S., Pardee, K., Collins, J. J. Synthetic biology devices for in vitro and in vivo diagnostics. Proceedings of the National Academy of Sciences of the United States of America. 112 (47), 14429-14435 (2015).
  2. Harbaugh, S. V., Goodson, M. S., Dillon, K., Zabarnick, S., Kelley-Loughnane, N.

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