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The protocol describes the formation of robust and biocompatible DNA-laden microcapsules as multiplexed in vitro biosensors capable of tracking several ligands.
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.
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....
1. Construction of plasmid vector.
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.......
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.......
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.
....Name | Company | Catalog Number | Comments |
(Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4 H)-one (DFHBI-1T) | Lucerna | DFHBI-1T | |
5x T4 DNA Ligase Buffer | ThermoFisher Scientific | 46300-018 | |
6x Blue Gel Loading Dye | New England BioLabs | B7021S | |
96-well plates, black circular | Corning | 3601 | |
Agarose | Sigma-Aldrich | A9539 | BioReagent, for molecular biology, low EEO |
Ampicillin sodium salt | Sigma-Aldrich | A0166 | powder or crystals, BioReagent, suitable for cell culture |
BlpI restriction enzymes | New England BioLabs | R0585S | |
Corning Disposable Vacuum Filter/Storage Systems | FisherScientific | 09-761-1 | |
Dimethyl sulfoxide, DMSO | Sigma-Aldrich | 472301 | ACS reagent, ≥99.9% |
DNA Plasmid, pET28c-F30-2x Broccoli (5.4 kb), BrocApt. | Addgene | Plasmid #66788 | |
DyLightTM550 Antibody Labeling kit (Invitrogen) | ThermoFisher Scientific | 84530 | |
E. coli S30 extract system for circular DNA | Promega | L1020 | |
Falcon Conical centrifuge tubes, 15 mL | FisherScientific | 14-959-53A | |
Falcon Conical centrifuge tubes, 50 mL | 14-432-22 | ||
Fisherbrand Microcentrifuge tubes, 1.5 mL | FisherScientific | 05-408-129 | |
Hydrofluoric acid, HF | Sigma-Aldrich | 695068 | ACS reagent, 48% |
Kanamycin sulfate | Sigma-Aldrich | 60615 | mixture of Kanamycin A (main component) and Kanamycin B and C |
KpnI restriction enzymes | New England BioLabs | R0142S | |
LB agar plate supplemented with 100 µg/mL ampicillin | Sigma-Aldrich | L5667 | pre-poured agar plates with 100 µg/mL ampicillin |
LB agar plate supplemented with 50 µg/mL kanamycin | Sigma-Aldrich | L0543 | pre-poured agar plates with 50 µg/mL kanamycin |
LB broth (Lennox grade) | Sigma-Aldrich | L3022 | |
Lithium bromide, LiBr | Sigma-Aldrich | 213225 | ReagentPlus, ≥99% |
Max Efficiency DH5-α competent E. coli strain | ThermoFisher Scientific | 18258012 | |
Methanol | MilliporeSigma | 322415 | anhydrous, 99.8% |
MilliQ-water | EMD MilliPore | Milli-Q Reference Water Purification System | |
MinElute PCR Purification Kit | Qiagen | 28004 | |
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, EDC | Sigma-Aldrich | E1769 | |
PBS (phosphate buffered saline) | ThermoFisher Scientific | 10010023 | 1x PBS, pH 7.4 |
Phusion High-Fidelity DNA Polymerase | New England Biolabs | M0530S | |
Polyethylenimine, branched | Sigma-Aldrich | 408727 | average Mw ~25,000 |
PURExpress In Vitro Protein Synthesis Kit | New England BioLabs | E6800S | |
QIAEX II Gel Extraction Kit | Qiagen | 20021 | |
QIAprep Spin Miniprep Kit | Qiagen | 27104 | |
Quick-Load 2-Log DNA Ladder (0.1-10.0 kb) | New England BioLabs | N0469S | |
SiO₂ silica microspheres, 4.0 µm | Polysciences, Inc. | 24331-15 | 10% aqueous solution |
Slide-A-Lyzer G2 Dialysis Cassettes, 3.5K MWCO, 15 mL | ThermoFisher Scientific | 87724 | |
Sodium carbonate, Na₂CO₃ | Sigma-Aldrich | 222321 | ACS reagent, anhydrous, ≥99.5%, powder |
Spectrum Spectra/Por Float-A-Lyzer G2 Dialysis Devices | FisherScientific | 08-607-008 | Spectrum G235058 |
SYBR Safe DNA gel stain | ThermoFisher Scientific | S33102 | |
T4 DNA Ligase (5 U/µL) | ThermoFisher Scientific | EL0011 | |
Theophylline | Sigma-Aldrich | T1633 | anhydrous, ≥99%, powder |
Tris Acetate-EDTA buffer (TAE buffer) | Sigma-Aldrich | T6025 | Contains 40 mM Tris-acetate and 1 mM EDTA, pH 8.3. |
UltraPure DNase/RNase-Free Distilled Water | FisherScientific | 10-977-023 | |
ZymoPURE II Plasmid MaxiPrep kit | ZymoResearch | D4202 |
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