加载编码RNA核酸适体和核糖开关的DNA质粒的多功能丝基微胶囊的制备
Summary
该协议描述了稳健且生物相容的富含DNA的微胶囊的形成，作为能够跟踪多种配体的多重体 外 生物传感器。
Abstract
我们介绍了一种通过牺牲球形核心上的逐层（LbL）组装方法制备富含DNA的丝素蛋白微胶囊的方案。在吸附质层和DNA质粒后，在单个蚕丝层的急性脱水过程中诱导蚕丝二级结构中的β片，促进了健壮微胶囊的形成。因此，分层是通过多次氢键和疏水相互作用发生的。在吸附多层壳后，核壳结构可以用金纳米颗粒（AuNPs）和/或抗体（IgG）进一步功能化，用于遥感和/或靶向递送。在二氧化硅核心上顺序沉积关键大分子的过程中调整几个关键参数，例如聚合物引物的存在、DNA 和丝蛋白的浓度以及许多吸附层，从而产生具有可变渗透性和 DNA 负载量的生物相容性、富含 DNA 的微胶囊。在二氧化硅核心溶解后，该方案证明了空心和坚固的微胶囊的形成，其中DNA质粒固定在胶囊膜的内表面。在DNA质粒和外部环境之间建立选择性渗透的生物相容性膜，在长期储存期间保存DNA，并在改善空间受限质粒的输出响应中发挥重要作用。在 体外 转录和翻译反应（无细胞系统）期间测试DNA模板的活性及其可及性。编码RNA发光适配体和核糖开关的DNA质粒被相应的分析物成功激活，如在壳膜中定位荧光标记的RNA转录物或GFPa1蛋白时可视化的那样。
Introduction
合成生物学领域通过利用微生物进化的自然机制来监测其环境和潜在威胁，为发展传感能力提供了独特的机会。重要的是，这些传感机制通常与保护这些微生物免受有害暴露的反应有关，调节基因表达以减轻负面影响或防止摄入有毒物质。人们已经付出了巨大的努力来设计这些微生物，以利用这些自然反应来创建全细胞传感器，但重新定向它们以识别新靶标和/或产生可测量的信号，该信号可用于定量目的（通常是荧光）1，2。目前，由于整个细胞或其某些遗传物质的泄漏，即使封装在聚合物基质中，对使用转基因微生物（GMO）的担忧，特别是在环境或人体中释放时，也表明需要利用这些传感方法的替代方法3。
利用基于微生物的传感的好处而不用担心转基因生物部署的一种强大方法是使用体外转录/翻译（IVTT）系统。从实践的角度来看，IVTT系统由含有大部分处于活性状态的细胞成分的混合物组成，这些成分是通过不同方式从细胞中“提取”的，包括超声处理，珠子跳动或其他方式4。该过程的最终产品是已经优化为执行转录和翻译的生化反应混合物，可用于以“开放容器”格式测试不同的传感器，而不受与使用整个细胞相关的限制（膜扩散、转化效率、细胞毒性等）。重要的是，可以定量添加不同的传感器组件，并通过不同的光学和光谱技术研究它们的影响，正如我们所展示的5。已经注意到，IVTT系统的性能可能不一致;然而，最近的研究已经显示了标准化其制备和表征的方法，这对于研究它们在传感器设计中的性能有很大帮助6。最近，已经证明了许多IVTT系统通过冻干纸基质中的组分来创建纸质测定的例子，包括检测重金属离子，药物，群体传感元件等7，8，9。基于IVTT的传感器的一个令人兴奋的应用领域是它们在不同类型的环境中的传感应用，包括土壤、水和人体。为了将这些IVTT系统部署到这些具有挑战性的环境中，需要实施一种封装方法来包含IVTT组件并保护它们免受降解。
IVTT系统最常见的封装方法包括使用脂质胶囊，胶束，聚合物体和其他紧密封闭的微容器10，11，12。这种方法的一个缺点是需要结合被动或主动机制来将材料运入和运出容器，以允许与外部环境通信并提供传感功能。为了克服其中的一些问题，本文报告了一种方法，该方法提供了一种简单而有效的方法来封装要在IVTT系统中表达的不同传感器设计的编码材料。该方法基于在目标质粒存在下使用生物聚合物的逐层（LbL）沉积来创建具有高孔隙率的空心微胶囊，这使得受保护的遗传物质与所选IVTT的不同组分相互作用。该研究表明，封装的质粒在该聚合物基质中激活时可以指导转录和翻译，如质粒编码的适配体和核糖开关对其相应靶标的响应所示。此外，这种LbL包被可在没有任何特殊储存条件的情况下保护质粒数月。
Protocol
1.质粒载体的构建。 通过扩增茶碱核糖开关 （ThyRS） 的编码序列与来自 pJ201：23976-RS-GFPa1 载体（由 DNA2.0 设计和创建）的 GFPa1 偶联并插入大 肠杆菌 表达载体 pSAL13，构建质粒载体 （pSALv-RS-GFPa1， 3.4 kb）。使用正向 （5′-CGTGGTACCGGTGGTGATGATACCCATCGTCTTGATG-3’） 和反向 （5′-CGTGCTCAGCTTAAGCCAGCTCGTAG-3’） 引物扩增 ThyRS 与 GFPa1 偶联的编码序列，并根据制造商的方案14</…
Representative Results
在这里，该研究解决了编码不同传感器设计（两种类型的RNA调控转录/翻译元件）的DNA模板在封装在丝蛋白胶囊中的功能。通过模板化逐层（LbL）组装制备关键组分的微胶囊：素数层，编码传感器设计的DNA质粒和丝素蛋白生物聚合物（图2）。以分层方式沉积大分子允许根据吸收层之间的分子间和内相互作用与壳的厚度来控制胶囊膜的渗透性。该系统的可调通透性提供了控制?…
Discussion
可以按照该协议制备装有各种类型的DNA编码传感器设计的选择性渗透水凝胶微胶囊。LbL方法的一个显着特点是能够在自下而上的组装过程中定制微胶囊的复杂性，这通常从牺牲模板上分子物质的吸附开始。通过仔细调整初始组分的浓度、pH条件和层数，可以制备具有不同DNA上样参数、功能和可调通透性的微胶囊23。为了增强胶囊的多功能性，可以使用AuNPs和IgG实现壳表面的进一步?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了空军科学研究办公室的LRIR 16RH3003J资助，以及美国国防部副部长办公室负责研究和工程的军事环境应用研究促进科技优先事项（ARAP）计划的支持。
ThyRS 的质粒载体序列 （pSALv-RS-GFPa1， 3.4 kb） 由 J. Gallivan 博士慷慨提供。 来自Bombyx mori 的蚕茧由马萨诸塞州塔夫茨大学的D.L. Kaplan博士慷慨捐赠。
Materials
| (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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Citazione di questo articolo
Drachuk, I., Harbaugh, S., Kelley-Loughnane, N., Chávez, J. L. Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches. J. Vis. Exp. (176), e62854, doi:10.3791/62854 (2021).