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 for quantification purposes (typically fluorescence)1,2. Currently, concerns with the use of genetically modified microorganisms (GMOs), especially when releasing in the environment or the human body, due to leakage of whole cells or some of their genetic material, even if encapsulated in a polymer matrix, suggest that alternative ways to exploit these sensing approaches are needed3.
A powerful approach to exploit the benefits of microorganisms-based sensing without the concern for the deployment of GMOs is the use of in vitro transcription/translation (IVTT) systems. From a practical perspective, IVTT systems consist of a mixture containing most of the cell components in an active state that has been "extracted" from cells by different means, including sonication, bead-beating, or others4. The final product of this process is a biochemical reaction mixture already optimized to perform transcription and translation that can be used to test different sensors in an "open vessel" format, without the constraints associated with the use of whole cells (membrane diffusion, transformation efficiency, cell toxicity, etc.). Importantly, different sensor components can be quantitatively added, and their effect studied by different optical and spectrometric techniques, as we have demonstrated5. It has been noticed that the performance of IVTT systems can be inconsistent; however, recent studies have shown approaches to standardize their preparation and characterization, which is of great help when studying their performance in sensor design6. Recently, many examples of IVTT systems using to create paper-based assays through the lyophilization of their components in paper matrices have been demonstrated, including detection of heavy metal ions, drugs, quorum sensing elements, and others7,8,9. An exciting application space for IVTT-based sensors is their use in sensing applications in different types of environments, including soil, water, and the human body. In order to deploy these IVTT systems to these challenging environments, an encapsulation approach need to be implemented to contain the IVTT components and protect them from degradation.
The most common encapsulation approaches for IVTT systems include the use of lipid capsules, micelles, polymersomes, and other tightly enclosed microcontainers10,11,12. One disadvantage of this approach is the need to incorporate either passive or active mechanisms to transport materials in and out of the containers to allow communication with the external environment and provide sensing capabilities. To overcome some of these issues, the study here reports a method that provides a simple yet effective approach to encapsulate the encoding materials for different sensor designs to be expressed in IVTT systems. This approach is based on the use of Layer-by-Layer (LbL) deposition of a biopolymer in the presence of the plasmids of interest to create hollow microcapsules with high porosity, which allows the protected genetic material to interact with the different components of the IVTT of choice. The study demonstrated that encapsulated plasmids could direct transcription and translation when activated within this polymeric matrix, as shown with the response of a plasmid-encoded aptamer and a riboswitch to their corresponding targets. Additionally, this LbL coating protects the plasmids for months without any special storage conditions.
1. Construction of plasmid vector.
2. Large-scale DNA purification.
3. Extraction of silk fibroin and preparation of initial materials.
4. Perform Layer-by-Layer deposition of a prime layer, DNA plasmids, and silk layers.
5. Dissolution of cores to obtain silk microcapsules.
6. Imaging of silk fibroin microcapsules using confocal laser scanning microscope (CLSM).
7. Estimation of the permeability of hollow microcapsules using molecular weight cut-off (MWCO) method.
8. In vitro activation of synthetic theophylline riboswitch in silk microcapsules
9. In vitro activation of broccoli aptamer in silk microcapsules
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 membrane based on inter- and intramolecular interactions between the absorbed layers and the thickness of the shell. The tunable permeability of the system offered the potential to control the diffusion of essential molecules while limiting the diffusion of large undesirable macromolecules through the capsule membrane20.
Homogeneous in size (4.5 µm) and robust silk fibroin microcapsules with a shell thickness of ~500 nm (20 layers) in a hydrated state can be produced when properly following the protocol (Figure 3)21. The LbL approach allowed to tune the loading capacity of DNA templates based on the initial concentration of the plasmids. The optimal DNA loading can be achieved by varying the initial concentration of DNA templates from 50-200 ng/µL. Table 1 represents the number of DNA copies retained in a single microcapsule upon completion of LbL encapsulation and was calculated for each sensor design based on the initial DNA concentrations and Mw of DNA plasmids according to Equation 1. The optimal DNA loading capacity was achieved with 32 and 20 DNA copies for ThyRS and BrocApt sensor designs, respectively. The assessment of loading capacity was important to have an accurate estimation of the encapsulated DNA concentrations to be able to titrate it during IVTT activation of the sensors by adding more concentrated capsule samples.
The permeability of the capsule shells can be systematically analyzed by following the MWCO method. The method estimates the pore size of the membrane based on the molecular weight of solute molecules that could not penetrate through the capsule membrane. By subjecting microcapsules to variable Mw fluorophore molecules and performing confocal imaging in the focal plane that corresponds to the largest diameter across multiple capsules, the permeability of the shell membranes can be assessed. ImageJ analysis allows to estimate the fluorescence intensities inside and outside of the capsules and identify fully permeable (outside and inside fluorophore intensities were comparable), partially permeable (50%-70% of capsules had lower fluorescence inside compared to outside), and not permeable (inside fluorophore intensity was lower compared to outside intensity) membranes (Figure 4). Converting Mw to hydrodynamic radius for each fluorophore allows estimating the mesh size of the capsule membrane (Table 2).
Selective permeability of protein capsules can be attained during systematic optimization of LbL deposition protocol by using variable concentrations of a prime layer (from 0-6 mg/mL), the concentration of a silk fibroin (from 0.5-2 mg/mL) and the number of deposited layers (from 10-25) until optimal permeability is achieved. In this protocol, optimal permeability between 25-32 nm was achieved with 6 mg/mL of PEI as a prime layer and 20 layers of 1 mg/mL of silk fibroin deposited during LbL assembly (Figure 4). This permeability range was ideal for the components of the IVTT system to percolate through the capsule shell. Typically, the largest molecular complexes of any IVTT system are prokaryotic ribosomal units, 30S and 50S, which, when bound by mRNA in a ribosomal complex, can reach up to ~20 nm in size22. The structure of the developed microcapsules shells resembles a highly intertwined network of silk fiber layers that are physically crosslinked by β-sheet blocks produced during LbL assembly. This membrane structure is semi-permeable: highly permeable to small molecules (e.g., ions, amino acids, peptides, sugars, etc.) and restricted to large molecules (e.g., high molecular weight proteins, glycoproteins, cells, etc.) that only allows percolation of molecules with a hydrodynamic radius less than 25 nm. Hence, silk fibroin microcapsules with optimal DNA loading and membrane permeability can be used as bioreactor carriers for IVTT activation.
The transcriptional and translational activity of ThyRS and BrocApt design sensors can be tested using commercially available IVTT systems. ThyRS requires translational activation of the reporter gene in the presence of theophylline ligand. Activation of synthetic ThyRS involves several steps to initiate gene expression, including mRNA synthesis, the conformational change of mRNA sequence upon binding to the analyte, release of the ribosome binding site, and the synthesis of the reporter GFPa1 protein. All these steps require free diffusion of the IVTT components through the capsule membrane. The proposed design of DNA-laden silk fibroin microcapsules improved the activation parameters of RNA-regulated sensor designs: Both the expression kinetics and fluorescence output were significantly higher in comparison to the free non-immobilized DNA (Figure 5A). The CLSM imaging also confirmed successful GFPa1 gene activation within capsules loaded with DNA plasmids encoding ThyRS sequences (Figure 5B–D).
Alternatively, the activation of BrocApt sensor design was carried out in the IVTT system that supports T7 E. coli promoter coupled transcription/translation in the presence of DHFBI-1T dye. The fluorescence output was initiated upon binding the fluorogenic dye to the mRNA sequence. Importantly, the output signal from silk microcapsules was several folds higher compared to non-encapsulated DNA samples of equivalent concentrations (Figure 6). Hence, silk fibroin microcapsules can retain the essential functionality of DNA-encoded sensor elements, which can be important for the design of new types of in vitro biosensor platforms for rapid and sensitive detection of analytes of interest.
Figure 1: Plasmid map and sequence of pSALv-RS-GFPa1. (A) The plasmid contains theophylline riboswitch (ThyRS) sequence placed upstream of GFPa1 encoding gene under the control of ptac promoter, β-lactamase (bla) encoding gene responsible for resistance to ampicillin. (B) The ptac promoter sequence is shown in bold and underlined; theophylline riboswitch sequence is shown in bold italic; GFPa1 encoding sequence is shown in bold; restriction sites sequences are underlined. Please click here to view a larger version of this figure.
Figure 2: Overview of the preparation of microcapsules using the Layer-by-Layer approach. Pristine SiO2 cores were firstly functionalized with PEI polymer, followed by the sequential deposition of DNA plasmids encoding different sensor designs and silk fibroin layers until the desired number of silk protein layers have been adsorbed. Hollow pristine microcapsules containing various DNA sensor designs can be produced by dissolving sacrificial cores. Activation of each biosensor design encapsulated in the microcapsule can be achieved during IVTT reactions in the presence of corresponding ligands. This figure has been reprinted from Drachuk, I. et al.21. Please click here to view a larger version of this figure.
Figure 3: Microscopy studies for DNA-loaded microcapsules. (A) Cross-sectional 2D and (B) Rendered 3D confocal microscopy images of hollow (SF)20 microcapsules loaded with DNA plasmids encoding ThyRS (32 DNA copies/capsule) and produced from 6 mg/mL PEI-primed SiO2 cores. Auto-fluorescence from silk fibroin capsules (DAPI channel, blue) and stained DNA (FITC channel, green) were applied to identify the localization of DNA plasmids and estimate the capsule membrane thickness. Inset in (A) represents intensity profiles for DNA and silk fibroin shells across the capsule. Scale bar: 2 µm. Inset in (B) represents the intensity profile for silk protein across the capsule shell. Membrane thickness was measured as the length at half intensity peak. Processing of the cross-sectional images and 3D rendering was performed using NIS imaging software on Nikon C2+ system. This figure has been reprinted from Drachuk, I. et al.21. Please click here to view a larger version of this figure.
Figure 4: Estimation of the membrane permeability for silk microcapsules using the MWCO method. Selective CLSM representative fluorescent images of hollow silk fibroin microcapsules subjected to FITC-Dextran solutions of various Mw (20 µM, diH2O). Capsules were prepared with a different number of layers and PEI concentrations. An increase in the concentration of a prime layer leads to increased colloidal stability of microcapsules with more permeable shells, while the elimination of the prime layer causes aggregation of capsules with less permeable shell membranes. Scale bar: 5 µm. This figure has been reprinted from Drachuk, I. et al.21. Please click here to view a larger version of this figure.
Figure 5: Activation of ThyRS sensor design in silk fibroin microcapsules. (A) Kinetics for GFPa1 expression during ThyRS activation from DNA plasmids loaded into 20-layered SF microcapsules (red-colored lines) and control non-encapsulated DNA (blue-colored lines). From top to bottom, concentrations of DNA in a sample volume (50 µL) were 6.5 ng/µL, 4.5 ng/µL, 2.5 ng/µL, and 0 ng/µL and correspond to the changes in color intensities. (B) CLSM images of silk fibroin capsules loaded with 32 DNA copies per capsule. Scale bar: 5 µm. (C) The image corresponds to cross-sectional intensity profiles corresponding to image (B) across two capsules (white line in B). The red line represents silk capsules, and the green line represents GFPa1 expressed in capsules. (D) The image represents rendered 3D silk capsules loaded with 32 copies of DNA after incubation in the IVTT system. Green fluorescence corresponds to the GFPa1 signal, and red fluorescence corresponds to fluorescently labeled silk layers. Scale bar: 2 µm. ThyRS activation was performed with theophylline (2 mM, DMSO) during the incubation of microcapsules in the IVTT system (S30 extract). This figure has been reprinted from Drachuk, I. et al.21. Please click here to view a larger version of this figure.
Figure 6: Activation of BroccApt in silk fibroin microcapsules. Comparison of transcription kinetics was performed for 20-layered silk microcapsules loaded with 30 DNA copies per capsule (red-colored lines) and control non-encapsulated DNA (blue-colored lines). From top to bottom, concentrations of encapsulated DNA in a sample were: 3.6 ng/µL, 2 ng/µL, 1 ng/µL and 0 ng/µL and correspond to the changes in color intensities. Concentrations of non-encapsulated DNA were: 20 ng/µL, 10 ng/µL, 5 ng/µL and 0 ng/µL and correspond to the changes in color intensities. Activation was performed with DHBFI-1T (100 µM, diH2O) during incubation in the PURE cell-free system. This figure has been reprinted from Drachuk, I. et al.21. Please click here to view a larger version of this figure.
DNA Sensor Design | Initial DNA Concentration (ng/µL) | Number of DNA copies per capsule (DNA/capsule) |
50 | 16 | |
ThyRS | 100 | 32 |
150 | 48 | |
200 | 64 | |
50 | 10 | |
BrocApt | 100 | 20 |
150 | 30 | |
200 | 40 |
Table 1: DNA copy number per capsule for each DNA design. The copy number was calculated according to Equation 1 and was correlated to the initial concentration of DNA plasmids, retention affinity of DNA sequences during the LbL deposition process, and the length of DNA plasmids.
Mw of FITC-Dextran (kDa) | Hydrodynamic Radius (nm) |
4 | 1.4 |
20 | 3.3 |
40 | 4.5 |
70 | 6 |
150 | 8.5 |
250 | 10 |
500 | 14 |
2,000 | 18 |
Table 2: Physical properties of FITC-dextrans. Hydrodynamic radius for each fluorophore was used to estimate the permeability of hollow silk fibroin microcapsules.
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 parameters, functionality, and tunable permeability can be prepared23. In order to amplify the versatility of the capsules, further functionalization of the shell surface with AuNPs and IgG can be achieved to implement biocompatible sensor carriers for in vivo diagnostics. Both of these alternative routes rely on the unique molecular structure of the silk fibroin. In situ reduction of Au3+ ions can be accomplished due to the presence of tyrosine residues (5.3% mol.) capable of reducing metal ions in the presence of an optimal reduction buffer. The conjugation of specific antibodies to the surface of AuNPs-functionalized protein microcapsules can be done via the implementation of carbodiimide activation chemistry. Both of these steps require careful development and optimization of the protocol in order to achieve the proper functionality of the biosensor capsules for future applications. For instance, to use these microcapsules as "smart" delivery systems, in which a molecular cargo is delivered upon specific stimuli (like pH or chemical cues), specific properties of these capsules should be characterized and tuned, including delivery efficiency, retention time, route of administration and disease model treatments.
The DNA-loaded microcapsules were characterized by the CLSM technique and fluorescence measurements. However, other characterization methods such as atomic force microscopy (AFM), cryogenic electron tomography (CEM), and direct stochastic reconstruction super-resolution microscopy (dSTORM) can be applied to differentiate specific structures of the microcapsules, including individual fibers, nanodomains, and localization of DNA plasmids24,25,26.
The developed protocol highlights the use of silk fibroin biopolymer as a biocompatible network material that preserves the tertiary structure of loaded DNA plasmids. The stability of immobilized DNA sequences within the silk fibroin network occurs via hydrophobic interactions and/or hydrogen bonding, which provide a protective microenvironment against pH inactivation, oxidation or hydrolysis27. In addition, the β-crystalline domains of silk fibroin provide a unique mechanical barrier, restricting the movement of entrapped macromolecules, and preventing DNA plasmids from degradation during storage in aqueous solutions.
The semi-permeable nature of silk fibroin microcapsules loaded with DNA templates created unique spatial and physicochemical conditions for the IVTT reactions. Transcriptionally and translationally activated RNA aptamer and riboswitch immobilized in silk fibroin microcapsules were able to produce an output signal that was at least three-fold higher in comparison to free non-immobilized sensors. In addition, the immobilization of DNA templates in microcapsules can significantly enhance the activity of encapsulated sensors beyond the detection limit of non-encapsulated ones. While non-encapsulated sensors had a minimal response at low DNA concentrations, the encapsulated sensors had a very distinct output response, which can be easily titrated to reflect the subtle changes in DNA concentrations. The improved response signal of encapsulated reporters was likely due to unrestricted diffusion of the components of IVTT machinery and reduced mobility of DNA plasmids. Several studies have recognized that the yield of protein synthesis in cell-free reactions is dependent upon the macromolecular environment28,29. Immobilization of DNA plasmids into silk network provided an effective confined environment restricting oligonucleotides movement and accelerating transcription/translation mechanism reactions even from several DNA copies21.
While the LbL method provides a very versatile approach to construct multifunctional assemblies in a controllable manner, the fabrication procedure is relatively time-consuming and typically requires processing adjustments to scale up the microcapsules yield. Another consideration in making biomolecule-based microcapsules is the compatibility of any given system with the requirement for the core dissolution after LbL deposition is completed. So far, to produce homogeneous in size and robust hollow silk-based DNA-laden microcapsules, the shells were deposited on sacrificial silica (SiO2) core templates, which required subsequent removal by hydrofluoric (HF) acid etching. Nor HF handling or etching are considered biocompatible processes, limiting their widespread use in practical applications. The alternative to SiO2 inorganic colloidal templates, carbonate cores are typically inert in forming complexes with polymer layers and can be dissolved under mild conditions using ethylenediaminetetraacetic acid (EDTA). However, adjusting sacrificial cores may affect the deposition properties of multilayers and the integrity of the shell structure, which requires further optimization of the protocol.
In summary, the current protocol allows the preparation of silk-based DNA-laden microcapsules with different sensor designs. The combination of a confined microenvironment provided by the LbL method and the use of silk fibroin as a biocompatible material improved the sensing properties of encapsulated DNA. With the rapid development of fluorogenic RNA aptamer technology, the potential use of DNA-laden silk microcapsules can be extended to multiplexed in vitro diagnostics. Recently, several variations of RNA-based fluorescent aptamers have emerged as powerful background-free technology for imaging RNA in live cells with high signal-to-noise ratio sensitivity30,31. By applying synthetic RNA nanotechnology to design artificial RNA aptamers, the fluorogenic properties of aptamers can be harnessed to detect specific ligands of interest. This technology will be specifically valuable to monitor biomarkers of interest in different formats, including point-of-use paper-based sensors, hydrogel-based patches for sweat or interstitial fluid, and implantable materials.
The authors have nothing to disclose.
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.
(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 |