使用CyanoGate模块化克隆工具包进行共生对蓝藻的遗传改造
Summary
在这里，我们提出一个协议，描述如何使用CyanoGate模块化克隆工具包组装自我复制载体，ii） 通过结合将载体引入蓝藻宿主，iii） 使用板读取器或流式细胞仪。
Abstract
蓝藻是一组不同的原核光合生物，可以进行基因改造，用于可再生生产有用的工业商品。合成生物学的最新进展导致开发若干克隆工具包，如CyanoGate，这是一个标准化的模块化克隆系统，用于建立质粒载体，以便随后转化或结合转移到蓝藻中。在这里，我们概述了组装自我复制载体（例如，携带荧光标记表达盒）和结合转移载体到蓝藻菌株Synechocystis sp. PCC 6803 或Synechococcus 的详细方法埃隆塔图斯UTEX 2973.此外，我们概述了如何使用板读器或流动细胞仪来描述遗传部件（例如启动器）的性能。
Introduction
蓝藻是自养菌，可用于各种天然和异物高价值代谢产物1、2、3、4、5的生物合成。 6.要扩大商业可行性，仍需要克服几个障碍，最显著的是，与异养生物平台（如大肠杆菌和酵母）相比，产量相对较低7。最近现有基因工程工具的扩展和合成生物学范式在蓝藻研究中的采用，正在帮助克服这些挑战，并进一步将蓝藻发展为高效的生物工厂89，10.
将DNA引入蓝藻的主要方法是转化、结合和电穿孔。通过转化或电穿孔转移到蓝藻的载体是”自杀”载体（即促进同源重组的综合载体），而自我复制的载体可以通过转化、联结或电穿孔。对于前者，一个协议可用于工程模型物种，适合自然转化11。最近，一种名为CyanoGate的蓝藻模块化克隆（MoClo）工具包已经开发出来，采用标准化的金门矢量组装方法，采用自然转化、电穿孔或结合12进行工程。
金门式装配技术近年来日益普及，装配标准和零件库现已可供各种生物使用，包括13、14、15、16 ， 17.金门使用IIS型限制酶（例如，BsaI、BpiI、BsmBI、BtgZI和AarI）和一套接受酶和独特的悬垂，以方便在”一锅”组装反应中对多个序列进行定向分层组装。IIS 型限制酶可识别独特的不对称序列，并切断其识别位点的固定距离，以生成交错的”粘性端”切口（通常为 4 核苷酸 [NT] 悬垂），随后可利用该切口驱动有序 DNA组装反应15，18。这促进了由通用语法（如 PhytoBricks 标准19）定义的模块化 0 级部件（例如启动器、打开的读取框架和终结器）的大型库的开发。然后，0 级部件可以容易地组装成 1 级表达式盒，然后更复杂的高阶组件（例如，多基因表达式构造）可以构建在选择12、15的接受器向量中。金门式装配技术的一个关键优势是，在高通量设施（如DNA铸造厂20、21）实现自动化，可以测试复杂的实验设计，不能轻易通过人工。
CyanoGate建立在已建立的工厂MoClo系统12，15。要将新部件合并到 CyanoGate 中，必须首先将零件序列进行驯化，即必须删除 BsaI 和 BpiI 的”非法”识别站点。对于开放读取帧的零件编码（即编码序列 CDS），识别站点可以通过在序列中生成同义词突变（即将协子更改为编码相同氨基酸残渣的替代方法）来中断识别位点。这可以通过多种方法实现，从DNA合成到聚合酶链式反应（PCR）扩增为基础的策略，如吉布森组装22。根据所使用的表达式主机，应注意避免引入可能抑制翻译效率的稀有科顿23。在启动器和终结器序列中移除识别站点通常是一项风险更大的工作，因为修改可能会影响功能，并且部件可能无法按预期执行。例如，对假定转录因子结合位点或启动剂内的核糖体结合位点的变化可能会改变对诱导/抑制的强度和响应能力。同样，对关键终结器结构特征（例如，GC富干、环和多U尾）的修改可能会改变终止效率和效应基因表达24，25。尽管可以使用多个在线资源来预测启动子和终止器序列的活动，并告知建议的突变是否会影响性能26和27，但这些工具通常不能预测在蓝藻28，29，30。因此，仍建议在体内对改性部件进行表征，以确认活性。为了协助克隆顽固的序列，CyanoGate包括基于BioBrick载体pSB4K512，16，31的低拷贝克隆接受载体。此外，爱丁堡基因组铸造厂还提供”设计和构建”门户，以帮助进行载体设计（dab.genomefoundry.org）。最后，也是最重要的，CyanoGate 包括两个 T 级接受载体设计（相当于 2 级接受载体）15，用于使用自杀载体将 DNA 引入蓝藻，或能够自我复制的广泛宿主范围载体在几个蓝藻菌种32，33，34。
在这里，我们将重点描述用于生成 T 级自我复制载体的协议，以及Synechocystis PCC 6803 和Synechocous UTEX 2973（Synechocystis PCC 6803 和 S. elongatus）的遗传修饰UTEX 2973 以后通过结合（也称为三亲交配）。细菌细胞之间的DNA的共生转移是一个描述良好的过程，以前曾用于工程蓝藻物种，特别是那些不能自然胜任的物种，如S.长龙UTEX 297335， 36，37，38，39，40，41.简而言之，用携带要转移的载体的大肠杆菌菌株（”货物”载体）和载体（在同一大肠杆菌菌株或附加菌株中）孵育，以促成结合（”动员器”和”帮助器”向量）。进行夫妻转移需要四个关键条件：1）参与DNA转移的细胞之间的直接接触，2）货物载体必须与结合系统兼容（即，它必须包含适当的转移来源（oriT），也称为bom（流动性基础）位点，3） DNA刻痕蛋白（例如，由生物基因编码），在oriT处刻痕 DNA 以启动 DNA 到青霉菌的单链转移，必须存在并表达从货物或帮手载体，和4）转移的DNA不得破坏在受体氰化细胞（即，必须抵抗降解，例如，限制内细胞内膜酶活性）35，42。使货物载体得以持续，复制的来源必须与受体青霉菌兼容，以便自我复制和扩散到子细胞后分裂。为了帮助条件3和4，几个帮助载体可以通过Addgene和其他商业来源，编码生物以及几个甲基酶，以保护从原生内分酶在宿主氰化酶43。在本协议中，分别由MC1061大肠杆菌菌株携带动员器和辅助载体pRK24（www.addgeneorg/51950）和pRL528（www.addgene.org/58495）促进结合。在选择用于夫妻转移的载体时，必须小心谨慎。例如，在 CyanoGate 套件中，自复制货物矢量 pPMQAK1-T 编码为 Mob 蛋白12。然而，pSEVA421-T不44，因此，生物必须从合适的帮助向量表达。使用的载体也应适合目标生物体。例如，在Anabaena sp. PCC 7120中，有效的夫妻转移需要一个帮助器载体，以保护动员器载体免受消化（例如，pRL623，它编码为三种甲基酶AvaiM，Eco47iiM和Ecot2iM）45， 46.
在本协议中，我们进一步概述了如何使用板读取器或流式细胞仪使用荧光标记来描述部件（即启动子）的性能。流量细胞计能够测量荧光在单个细胞的基础上为大量。此外，流量细胞计允许用户”门”获取的数据并消除背景噪音（例如，从培养物或污染中的颗粒物中）。相反，板读取器获取给定培养量的聚合荧光测量，通常在几个复制孔中。与细胞仪不同，板式读片器的主要优势包括成本更低、可用性更高，并且通常不需要用于下游数据分析的专业软件。板读机的主要缺点是与细胞仪相比灵敏度相对较低，并且与测量培养物的光学密度存在潜在问题。对于比较分析，必须对每口孔的板读取器样本进行标准化（例如，对培养密度进行测量，通常以 750 nm [OD750] 时的光密度为吸光度），这可能导致样品的精度也致密和/或未很好地混合（例如，当容易聚集或絮凝时）。
作为概述，这里我们详细介绍了生成 0 级零件的原则，然后使用 CyanoGate 套件进行分层组装，并将克隆到适合夫妻转移的载体中。然后，我们演示了夫妻转移过程，选择表达荧光标记的轴跨结子菌株，以及随后使用流式细胞仪或板读取器获取荧光数据。
Protocol
Representative Results
Discussion
与其他矢量装配方法相比，金门装配具有几个优点，特别是在可伸缩性20，21方面。然而，在实验室中建立金门系统需要时间来熟悉各个部件和接受方矢量库以及整体装配流程。对于更复杂的程序集或并行执行大量复杂程序集（例如，制作一套包含多个基因表达盒的 T 级矢量），通常需要仔细规划。我们建议首先列出所需的所有基因表达盒组合，然后映?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢PHYCONET生物技术和生物科学研究理事会（BBSRC）工业生物技术和生物能源网络（NIBB）和工业生物技术创新中心（IBioIC）的财政支持。GARG、AASO 和 AP 感谢 BBSRC EASTBIO CASE 博士计划（授权编号 BB/M010996/1）、国家技术与技术委员会 （CONACYT） 博士计划以及 IBioIC-BBSRC 协作培训伙伴关系 （CTP） 博士计划的资助支持，分别。我们感谢康拉德·穆利诺（伦敦玛丽女王大学）和波尔·埃里克·詹森和朱莉·安妮玛丽·齐塔·泽德勒（哥本哈根大学）对质粒病媒和协议的贡献和建议。
Materials
| 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) | Thermo Fisher Scientific | R0404 | Used in 2.1.3. |
| Adenosine 5′-triphosphate (ATP) disodium salt | Sigma-Aldrich | A2383 | Used in Table 2. |
| Agar (microbiology tested) | Sigma-Aldrich | A1296-500g | Used in 8.3. |
| Agarose | Bioline | BIO-41026 | Used in 6. |
| Attune NxT Flow Cytometer | Thermo Fisher Scientific | – | Used in 4.3.1. |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A2153 | Used in Table 2. |
| BpiI (BbsI) | Thermo Fisher Scientific | ER1011 | Used in Table 2. |
| BsaI (Eco31I) | Thermo Fisher Scientific | ER0291 | Used in Table 2. |
| Carbenicillin disodium | VWR International | A1491.0005 | Used in 2.1.3. |
| Corning Costar TC-Treated flat-bottom 24 well plates | Sigma-Aldrich | CLS3527 | Used in 4.1.3. |
| Dimethyl Sulfoxide (DMSO) | Thermo Fisher Scientific | BP231-100 | Used in 3.6.2. |
| DNeasy Plant Mini Kit | Qiagen | 69104 | DNA extraction kit. Used in 5. |
| FLUOstar Omega Microplate reader | BMG Labtech | – | Used in 4.2.2. |
| GeneJET Plasmid Miniprep Kit | Thermo Fisher Scientific | K0503 | Plasmid purification kit. Used in step 2.3.2. |
| Glass beads (0.5 mm diameter) | BioSpec Products | 11079105 | Used in 5.2. |
| Glycerol | Thermo Fisher Scientific | 10021083 | Used in 2.3.1, 3.6.2. |
| Isopropyl-beta-D-thiogalactopyranoside (IPTG) | Thermo Fisher Scientific | 10356553 | Used in 2.1.3. |
| Kanamycin sulphate (Gibco) | Thermo Fisher Scientific | 11815-024 | Used in 2.1.3. |
| Membrane filters (0.45 μm) | MF-Millipore | HAWP02500 | Used in 3.3.7 |
| Microplates, 96-well, flat-bottom (Chimney Well) µCLEAR | Greiner Bio-One | 655096 | Used in 4.2.1. |
| Monarch DNA Gel Extraction Kit | New England Biolabs | T1020S | Used in 1.1.2.2. |
| Monarch PCR DNA Cleanup Kit | New England Biolabs | T1030 | DNA purification kit. Used in 1.1.2.3. |
| Multitron Pro incubator with LEDs | Infors HT | – | Shaking incubator with white LED lights. Used in 4.1.4. |
| MyTaq DNA Polymerase | Bioline | BIO-21108 | Used in 7.1. |
| NanoDrop One | Thermo Fisher Scientific | ND-ONE-W | Used in 2.3.3. |
| One Shot TOP10 chemically competent E. coli | Thermo Fisher Scientific | C404010 | Used in 2.1.1. |
| Phosphate buffer saline (PBS) solution (10X concentrate) | VWR International | K813 | Used in 4.3.2. |
| Q5High-Fidelity DNA Polymerase | New England Biolabs | M0491S | Used in 1.1.2.1. |
| Quick-Load 1 kb DNA Ladder | New England Biolabs | N0468S | Used in Figure 4. |
| Screw-cap tubes (1.5 ml) | Starstedt | 72.692.210 | Used in 3.6.3 |
| Spectinomycin dihydrochloride pentahydrate | VWR International | J61820.06 | Used in 2.1.3. |
| Sterilin Clear Microtiter round-bottom 96-well plates | Thermo Fisher Scientific | 612U96 | Used in 4.3.1. |
| T4 DNA ligase | Thermo Fisher Scientific | EL0011 | Used in Table 2. |
| TissueLyser II | Qiagen | 85300 | Bead mill. Used in 5.2. |
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