作者感谢Paul Hooykaas教授提供荷兰莱顿大学莱顿生物研究所的pCAMBIA1302载体和 根癌农杆 菌AGL1。作者还要感谢Eva Colic在培养荧光转化体方面的帮助。这项工作由加拿大自然科学与工程研究委员会和Mitacs Accelerate计划资助。

除非另有说明，否则所有培养基和溶液必须在使用前进行高压灭菌。所有离心管、移液器吸头等在使用前应无菌或高压灭菌。为了便于参考， 表1中列出了该协议中使用的培养基配方。
1. 根癌曲霉电 感受态细胞的制备
<ol><li>将农杆菌（AGL-1）接种到25mL无菌摇瓶中，该瓶装有LB培养基（补充利福平，20mg / L - 1），来自冷冻甘油原液，并在28-30°C和150rpm下生长过夜。 注： 农杆菌 菌株AGL-1对利福平、氨苄西林、氯霉素和链霉素具有抗性（见 材料表），可从多家供应商处获得。</li>
	<li>第二天，通过将0.5μL过夜培养物加入50mL高压灭菌的超纯水中，将培养物稀释1,00,000倍，并将10μL稀释的培养物铺在LB板上，然后在28-30°C下孵育1-3天。</li>
	<li>选择一个菌落，并在28-30°C下用利福平在LB中开始20mL过夜培养。</li>
	<li>第二天，在装有9 mL过夜培养物的摇瓶中接种500 mL LB培养基（无抗生素），并在28-30°C下生长，直到OD600 达到0.5。</li>
	<li>将培养物均匀地分成10个50mL离心管，并在冰上冷却30分钟。将离心机预冷至4°C。</li>
	<li>将试管在4°C和4000× g 离心15分钟。随后，使用移液管除去尽可能多的上清液，并将每个试管中的沉淀重悬于 50 mL 冰冷水中。</li>
	<li>将细胞在4°C和4000× g 离心15分钟。除去上清液，将沉淀重悬于每个试管中 25 mL 冰冷的水中。</li>
	<li>将细胞在4°C和4000× g 离心15分钟。除去上清液，将沉淀重悬于每个试管中 1 mL 冰冷的 10% （w/v） 甘油中。</li>
	<li>将所有试管合并到一个50mL试管中，并将细胞在4°C和4000× g 下离心15分钟。除去上清液，将沉淀重悬于400μL冰冷的10%（w / v）甘油中。细胞浓度应约为 1-3 x10 10 个细胞/mL。</li>
	<li>将细胞以 50 μL 等分试样的形式分布在无菌预冷微管中。在液氮中冷冻并储存在-80°C冰箱中。</li>
</ol>2. 根癌不动杆菌的电穿孔
<ol><li>将 50 μL AGL-1 根癌曲霉 电感受态细胞加入预冷的 0.1 mm 比色皿中，并加入 2 μL pCAMBIA1302 或类似质粒 （浓度 15 ng/μL）（参见 材料表）。</li>
	<li>使用电穿孔仪在 2400 V 下电穿孔（200 Ω，电容扩展器 250 μFD，电容 25 μFD，指数衰减，参见 材料表）。时间常数应为 >4.5 ms，才能成功进行指数衰减电穿孔。</li>
	<li>立即向比色皿中加入 1 mL LB 培养基，轻轻上下移液以混合。将1mL重悬细胞转移到1.5mL微管中，并在28-30°C下孵育至少1小时以恢复。</li>
	<li>将细胞铺在含有适当抗生素的LB琼脂上（即，50mg / L卡那霉素用于pCAMBIA1302的原核选择）。</li>
	<li>在28-30°C孵育2-3天。</li>
	<li>选择一个菌落，用适当的选择（50mg / L卡那霉素用于pCAMBIA1302）在LB中生长过夜，并在-80°C下使用50%甘油冷冻保存含质粒的菌株以备将来使用。</li>
</ol>3. 寻常梭菌的AMT
注：同时制备寻常念珠菌（UTEX 395，见材料表）和根癌农杆菌培养物进行共培养。寻常隐杆菌的培养应在制备根癌隐杆菌培养物前 3 天开始。根据 Kumar 等人发表的协议进行了修改7。
<ol><li> 准备寻常梭菌培养物<ol><li>寻常梭菌 传统上储存在斜面上或在光照条件下保持在对数期培养物中。从OD600 = 1.0的对数期培养物开始，将0.5mL扩散到Tris-乙酸盐 - 磷酸盐（TAP，参见表1）琼脂平板（ 1.2%w / v琼脂A）上，并在25°C下在光照下生长5天（50μE/ m2秒）。这将产生大约 5 x 106 个细胞。</li>
	</ol></li>
	<li>准备根癌不动杆菌培养物 <ol><li>通过使用甘油储备液接种补充有 15 mM 葡萄糖、20 mg/L 利福平和 50 mg/L 卡那霉素的 10 mL LB 培养基，在摇床中培养 根癌曲 霉菌 AGL-1 pCAMBIA1302 菌株（参见 材料表）。在28-30°C和250rpm下孵育过夜。</li>
		<li>将 1 mL 过夜培养物接种到 50 mL LB 中，加入 15 mM 葡萄糖、20 mg/L 利福平和 50 mg/L 卡那霉素，并在 28-30 °C 和 250 rpm 下孵育直至 OD600 达到 1.0。</li>
	</ol></li>
	<li>共培养寻常念珠菌和根癌曲霉
	<ol><li>为了准备用于共培养的 根癌不动杆菌 培养物，将生长的培养物转移到 50 mL 管中，并在室温下以 4000 x g 离心 30 分钟。使用移液管除去上清液，并用诱导培养基洗涤细胞两次。 注：使用的诱导培养基是调节至pH 5.5的TAP培养基，并补充有F/2培养基痕量金属和含有200μM乙酰丁香酮的维生素（AS，参见 材料表）。将细胞重悬于诱导培养基中，使OD600 = 0.5。</li>
		<li>为了制备用于共培养的寻常念珠菌培养物，将 25 mL 诱导培养基加入到含有 ~ 5 x 106 个细胞的寻常梭菌平板中。转移到 50 mL 试管中。在室温下以4000×g离心细胞15分钟，弃去上清液。</li>
		<li>将藻类细胞沉淀物与200μL细菌悬浮液（OD600：0.5）混合，并在21-25°C，150rpm的旋转振荡器中孵育1小时。</li>
		<li>将混合培养物（200μL）铺展到补充有15mM葡萄糖的诱导培养基板上，并在21-25°C下在黑暗中孵育3天。</li>
		<li>3天后，使用补充有400mg / L头孢噻肟或20mg / L四环素（参见材料表）的10mLTAP培养基收集微藻，并在21-25°C的黑暗中孵育2天，以消除培养物中的根癌杆菌。 </li>
		<li>将 500 μL 培养物铺在选择性培养基上，例如，补充有 20-70 mg/L 潮霉素 B（使用 pCAMBIA1302 时）和 400 mg/L-1 头孢噻肟或 20 mg/L 四环素的 TAP 培养基。在黑暗中孵育21-25°C2天，然后将它们放入照明室中。 注意：耐药菌落将在光照后 5-7 天出现。</li>
		<li>从转化平板中挑选单个菌落，并在补充有400mg / L头孢噻肟或20mg / L四环素和20-70mg / L潮霉素B的TAP琼脂平板上重新划线。</li>
	</ol></li>
</ol>4. 菌落PCR（cPCR）确认 寻常梭菌 转化体中的基因整合
<ol><li>使用植物基因组 DNA 提取试剂盒从单个菌落中传代培养至少两次后，从寻常梭菌转化体中提取基因组 DNA（参见材料表）。以此为PCR模板，设计T-DNA的mgfp5区域引物（mgfp5-Fwd：5'CCCATCTCATAAATAACGTC 3'和M13-Rev：5'CAGGAAACAGCTATGAC 3'）。<ol><li>使用 Q5 高保真 DNA 聚合酶预混液试剂盒（参见 材料表），进行聚合酶链反应 （PCR） 以验证转基因整合。 注：本研究使用以下条件：98°C3分钟;35 次循环（98 °C 持续 10 秒，57 °C 持续 30 秒，72 °C 持续 1 分钟）;72°C5分钟。使用 pCAMBIA1302 作为阳性对照，使用野生型 寻常念珠菌 基因组 DNA 作为阴性对照。</li>
	</ol></li>
	<li>为了确认样品中没有pCAMBIA1302，由于 根癌曲霉的残留污染，使用菌落PCR。将少量转化细胞加入10μL无菌水中，并在98°C下煮沸15分钟。将其用作 PCR 的模板。<ol><li>在pCAMBIA1302（B1-Fwd：5'AGTAAAGGAGAAGAACTTTTC 3'和B1-Rev：5'CCTGATGCGGTATTTTCTC 3'）上设计T-DNA以外的区域引物。 注：本研究使用以下条件：94°C3分钟;30 次循环（94 °C 持续 30 秒，48 °C 持续 30 秒，68 °C 持续 5 分钟）;68°C7分钟使用煮沸的 根癌曲 霉AGL-1（pCAMBIA1302）菌落作为阳性对照，使用煮沸的 野生型寻常梭菌菌 落作为阴性对照。</li>
	</ol></li>
	<li>为确认样本中不存在 A. tumerfaciens 污染，请使用菌落 PCR。将少量转化细胞加入10μL无菌水中，并在98°C下煮沸15分钟。将其用作 PCR 的模板。<ol><li>设计AGL-1毒力质粒（virE2-Fwd：5'AGGGAGCCCTACCCG 3'和virE2-Rev：5'GAACCAGCCTGGAGTTCG 3'）上所含virE2基因的引物。 注：本研究使用以下条件：94°C3分钟;30 次循环（94 °C 持续 30 秒，53 °C 持续 30 秒，68 °C 持续 3 分钟）;68°C7分钟使用煮沸的 根癌曲 霉AGL-1（pCAMBIA1302）菌落作为阳性对照，使用煮沸的 野生型寻常梭菌菌 落作为阴性对照。</li>
	</ol></li>
	<li>在DNA琼脂糖凝胶和分子量标准（1 Kbp DNA分子量标准，参见 材料表）上运行PCR样品，以确认所得片段的大小16。</li>
</ol>5. 测量转化体的荧光
<ol><li>将来自对数期维持培养物或TAP琼脂斜的每种转化体接种到补充有200mg / L - 1头孢噻肟和25mg / L - 1潮霉素B的50mLTAP中，使起始OD600 = 0.1。用野生型寻常型C. várgaris和仅200mg / L-1头孢噻肟作为阴性对照接种一种培养物。在25°C下以150rpm与150μmol/ m2秒的光合有效光一起孵育（参见材料表）。</li>
	<li>每 24 小时，取 300 μL 样品，并在 96 孔板读数仪或紫外/可见光谱仪和荧光计中测量吸光度和荧光（激发 488 nm，发射 526 nm）（参见 材料表）。使用底部透明的黑色板进行同时测量。</li>
</ol>6. 粗蛋白提取、蛋白纯化、SDS-PAGE电泳
<ol><li>将转化体 （#50） 从对数期维持培养物接种到 300 mL 的 TAP 中，补充有 200 mg/L-1 头孢噻肟和 25 mg/L-1 潮霉素 B，以达到 OD680 = 0.1。用野生型寻常型C. várgaris和仅200mg / L-1头孢噻肟作为阴性对照接种一种培养物。在25°C下以150rpm孵育150μmol/ m2秒的光合活性光。</li>
	<li>使用 5 mL 裂解缓冲液（见 表 1）从转化体 （#50） 和野生型菌株中提取粗蛋白样品，然后超声处理 4 分钟。</li>
	<li>用Ni-NTA树脂通过5 mL聚丙烯色谱柱纯化粗蛋白样品（参见 材料表）。</li>
	<li>冻干 15 mL 纯化的蛋白质样品，并将其重悬于 1 mL 裂解缓冲液中用于 SDS-PAGE 分析17。</li>
</ol>

利用根癌农杆菌在小球藻中的稳定基因整合

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R., Fuqua, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+maintenance+of+Agrobacterium.">Laboratory maintenance of Agrobacterium.</a> Current Protocols in Microbiology. Chapter 1: Unit 3D.1. , (2012).</li><li>Haddadi, F., Abd Aziz, M., Abdullah, S. N., Tan, S. G., Kamaladini, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+Agrobacterium-mediated+transformation+of+strawberry+cv.+Camarosa+by+a+dual+plasmid+system.">An efficient Agrobacterium-mediated transformation of strawberry cv. Camarosa by a dual plasmid system.</a> Molecules. 20 (3), 3647-3666 (2015).</li><li>Wang, X., Ryu, D., Houtkooper, R. H., Auwerx, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+use+and+abuse:+a+threat+to+mitochondria+and+chloroplasts+with+impact+on+research,+health,+and+environment.">Antibiotic use and abuse: a threat to mitochondria and chloroplasts with impact on research, health, and environment.</a> Bioessays. 37 (10), 1045-1053 (2015).</li><li>Gelvin, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+DNA+repair+and+Agrobacterium+T-DNA+integration.">Plant DNA repair and Agrobacterium T-DNA integration.</a> International Journal of Molecular Sciences. 22 (16), 8458 (2021).</li></ol>

没有宣布任何利益冲突。

转换效率与几个不同的参数相关联。用于AMT的 根癌不动杆菌菌 株的选择至关重要。AGL-1 是已发现的最具侵袭性的菌株之一，因此，已常规用于植物 AMT。 用葡萄糖 （15-20 mM） 补充诱导培养基对于 AMT 效率也很重要。考虑到 寻常梭菌 可以在光养和异养条件下生长，微藻培养基中通常省略葡萄糖或其他碳源以防止污染。例如，Bold 的基础培养基 （BBM） 是 C. vulgaris19<...

根癌农杆菌是一种革兰氏阴性土壤传播细菌，具有独特的界间基因转移能力，因此被誉为“天然基因工程师”1。这种细菌可以通过 IV 型分泌系统将 DNA （T-DNA） 从肿瘤诱导质粒 （Ti-Plasmid） 转移到宿主细胞中，导致 T-DNA 在宿主基因组内的整合和表达 1,2,3,4。在自然环境中，这个过程会导致植物肿瘤形成，通常称为冠瘿病。然而，农杆菌也可以将 T-DNA 转移到各种其他生物体中，包括酵母、真菌、藻类、海胆胚胎，甚至在实验室条件下的人类细胞 5,6,7,8。
利用这一自然系统， 根癌农杆菌介导的转化 （AMT） 通过修饰 Ti 质粒的 T-DNA 区域，将目标基因随机整合到宿主细胞的核基因组中。为此，广泛使用的AMT植物表达载体是pCAMBIA13029。研究人员可以在 大肠杆 菌中采用简单的克隆工作流程，然后将所需载体转移到 根癌不杆菌 中，以便随后转移到目标宿主中。
绿色微藻是真核生物，与陆地植物有许多相似之处，但对基因改造非常顽固。然而，遗传转化在微藻的基础和生物技术研究中都起着至关重要的作用。在几种微藻物种中，特别是莱茵衣藻，通过 AMT 的遗传转化已成功引入转基因，例如人白细胞介素 2 （hIL-2）、严重急性呼吸系统综合症冠状病毒 2 受体结合域 （SARS-CoV-2 RBD） 和两种抗菌肽 （AMP）10、11、12、13。其中，小球藻是一种不那么挑剔和快速生长的绿藻物种，在碳水化合物、蛋白质、营养保健品、色素和其他高价值化合物的可持续生产方面具有巨大的潜力14。然而，缺乏可靠的工具来创造转基因的寻常梭菌菌株阻碍了其商业进展。由于在 C. vulgaris15 中使用 AMT 的已发表作品数量有限，并且鉴于植物和微藻培养之间存在相当大的差异，因此优化 AMT 协议变得至关重要。
在这项研究中，研究人员在花椰菜花叶病毒（CamV）35S启动子的下游插入绿色荧光蛋白（mGFP5），并添加了组氨酸标签，将其用作蛋白质表达的报告基因。使用潮霉素B选择转化体，经过20代以上的传代培养，转化保持稳定。本研究中使用的pCAMBIA1302质粒可以很容易地适应任何目的基因。此外，所提供的方法和材料可以针对具有活性 CamV35S 启动子的其他绿藻物种进行调整，因为该启动子用于潮霉素选择。

<table><tbody><tr><td>1 Kb Plus DNA ladder</td><td>FroggaBio</td><td>DM015</td><td></td></tr><tr><td>Acetosyringone</td><td>Fisher Scientific</td><td>D26665G</td><td></td></tr><tr><td>&lt;em&gt;Agrobacterium tumefaciens&lt;/em&gt;</td><td>Gold Biotechnologies</td><td>Strain: AGL-1; Gift from Prof. Paul Hooykaas</td><td>Genotype: C58 RecA (Rif&lt;sup&gt;R&lt;/sup&gt;/Carb&lt;sup&gt;R&lt;/sup&gt;) pTiBo542DT-DNA</td></tr><tr><td>Biotin</td><td>Enzo Life Sciences</td><td>89151-400</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>VWR</td><td>BDH9224-1KG</td><td></td></tr><tr><td>Cefotaxime</td><td>AK Scientific</td><td>J90010</td><td></td></tr><tr><td>&lt;em&gt;Chlorella vulgaris&lt;/em&gt;</td><td>University of Texas at Austin Culture Collection of Algae</td><td>Strain: UTEX 395</td><td>Wildtype strain</td></tr><tr><td>CoCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>C8661-25G</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>EMD Millipore</td><td>CX2185-1</td><td></td></tr><tr><td>FeCl&lt;sub&gt;3&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>VWR</td><td>BDH9234-500G</td><td></td></tr><tr><td>Gene Pulser Xcell Electroporator</td><td>Bio-Rad</td><td>1652662</td><td>Main unit equipped with PC module.</td></tr><tr><td>GeneJET Plant Genome Purification Kit</td><td>Thermo Scientific</td><td>K0791</td><td></td></tr><tr><td>Glacial acetic acid</td><td>VWR</td><td>CABDH3093-2.2P</td><td></td></tr><tr><td>Glycerol</td><td>BioBasic</td><td>GB0232</td><td></td></tr><tr><td>HEPES Buffer</td><td>Sigma Aldrich</td><td>H-3375</td><td></td></tr><tr><td>Hygromycin B</td><td>Fisher Scientific</td><td>AAJ6068103</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>VWR</td><td>BDH9266-500G</td><td></td></tr><tr><td>Kanamycin</td><td>Gold Biotechnologies</td><td>K-250-25</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>VWR</td><td>BDH9268-500G</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>VWR</td><td>97062-134</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>JT Baker</td><td>BAKR2540-01</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;</td><td>VWR</td><td>BDH7971-1</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;EDTA&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>JT Baker</td><td>8993-01</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&amp;middot;&lt;/sub&gt;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>JT Baker</td><td>BAKR3764-01</td><td></td></tr><tr><td>NaCl</td><td>VWR</td><td>BDH7257-7</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Millipore Sigma</td><td>CA80058-650</td><td></td></tr><tr><td>NaNO&lt;sub&gt;3&amp;nbsp;&lt;/sub&gt;</td><td>VWR</td><td>BDH4574-500G</td><td></td></tr><tr><td>NEBExpress Ni Resin</td><td>NewEngland BioLabs</td><td>NEB #S1427</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>VWR</td><td>BDH9208-500G</td><td></td></tr><tr><td>pCAMBIA1302</td><td>Leiden University</td><td>Gift from Prof. Paul Hooykaas</td><td>pBR322, Kan&lt;sup&gt;R&lt;/sup&gt;, pVS1, T-DNA(CaMV 35S/Hyg&lt;sup&gt;R&lt;/sup&gt;/CaMV polyA, CaMV 35S promoter/mgpf5-6xhis/NOS terminator)</td></tr><tr><td>Polypropylene Columns (5 mL)</td><td>QIAGEN</td><td>34964</td><td></td></tr><tr><td>Precision Plus Protein Unstained Protein Standards, Strep-tagged recombinant, 1 mL</td><td>Bio-Rad</td><td>1610363</td><td></td></tr><tr><td>Rifampicin</td><td>Millipore Sigma</td><td>R3501-1G</td><td></td></tr><tr><td>SunBlaster LED Strip Light 48 Inch&amp;nbsp;</td><td>SunBlaster</td><td>210000000906</td><td></td></tr><tr><td>Synergy 4 Microplate UV/Vis spectrometer&amp;nbsp;</td><td>BioTEK</td><td>S4MLFPTA</td><td></td></tr><tr><td>Tetracycline</td><td>Thermo Scientific Chemicals</td><td>CAAAJ61714-14</td><td></td></tr><tr><td>TGX Stain-Free FastCast Acrylamide Kit, 12%</td><td>Bio-Rad</td><td>1610185</td><td></td></tr><tr><td>Thiamine</td><td>TCI America</td><td>T0181-100G</td><td></td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP152-500</td><td></td></tr><tr><td>Tryptone</td><td>BioBasic</td><td>TG217(G211)</td><td></td></tr><tr><td>Vitamin B12 (cyanocobalamin)</td><td>Enzo Life Sciences</td><td>89151-436</td><td></td></tr><tr><td>Yeast Extract</td><td>BioBasic</td><td>G0961</td><td></td></tr><tr><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>JT Baker</td><td>4382-01</td><td></td></tr></tbody></table>

agrobacterium tumefaciens-mediated genetic engineering of green microalgae, chlorella vulgaris

根癌农杆菌介导的转化（AMT）是一种广泛使用的植物基因组操纵工具。然而， 根癌曲霉 表现出将基因转移到多种物种的能力。许多微藻物种缺乏将目标基因可靠地整合到其核基因组中的成熟方法。为了利用微藻生物技术的潜在优势，简单高效的基因组操作工具至关重要。本文，利用报告绿色荧光蛋白（mGFP5）和潮霉素B的抗生素耐药性标记物，提出了针对工业微藻物种 寻常小球藻的优化AMT方案。经过十代以上的传代培养后， 通过荧光定量 mGFP5 的表达，表明 T-DNA 盒的稳定转化。该方案允许在两周内可靠地生成多个转基因 寻常念珠 菌菌落，采用市售的pCAMBIA1302植物表达载体。

为了显示使用上述方法成功转化，将寻常梭菌与含有pCAMBIA1302质粒的AGL-1或不使用质粒（野生型并接种在补充有潮霉素B和头孢噻肟的TAP琼脂上）共培养（图1A）。最左边的板显示了能够在潮霉素 B/头孢噻肟平板上生长的转化菌落，中间板显示野生型 AGL-1 不能在潮霉素 B/头孢噻肟平板上生长。最右边的板显示，当不使用选择（仅头孢噻肟）时，寻常梭菌转化体和?...

Watch this Scientific Journal Video about Optimized Transformation Protocol for Chlorella vulgaris Using Agrobacterium tumefaciens at JoVE.com

作者聚焦：使用根癌农杆菌优化寻常小球藻的转化方案 

该方案概述了 利用根癌农杆菌介导的转化（AMT）将目的基因整合到绿色微藻 小球藻的核基因组中，从而产生稳定的转化体。

根癌农杆菌介导的绿色微藻、寻常小球藻的基因工程

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit ...

研究范围集中在开发和优化寻常小球藻转化方案上。我们的目标是使用根癌农杆菌和特定质粒 pCAMBIA1302 创建稳定的转基因小球藻菌株，使他们能够插入目标基因和生物技术应用的潜力。代谢工程和合成生物学、显微镜和成像、CRISPR-Cas9基因编辑、生物信息学、计算建模、遥感和卫星成像、纳米技术、生物加工和下游加工。本研究介绍了一种使用特定质粒和标记物对微藻小球藻进行优化的根癌农杆菌培养基转化方案。它为通过技术增强微藻奠定了基础，克服了以前在可靠的寻常梭菌转化工具方面的局限性。

agrobacterium-tumefaciens-mediated-genetic-engineering-green

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris

3.1K 次观看.  University of Waterloo. 研究范围集中在开发和优化寻常小球藻转化方案上。我们的目标是使用根癌农杆菌和特定质粒 pCAMBIA1302 创建稳定的转基因小球藻菌株，使他们能够插入目标基因和生物技术应用的潜力。代谢工程和合成生物学、显微镜和成像、CRISPR-Cas9基因编辑、生物信息学、计算建模、遥感和卫星成像、纳米技术、生物加工和下游加工。本研究介绍了一种使用特定质粒和标记物对微藻?...

视频: 作者聚焦：使用根癌农杆菌优化寻常小球藻的转化方案 

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

心肌组织工程生物衍生的注射材料的制备

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

科研

生物工程

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

龙工程的程序

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

小鼠模型肠组织工程

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

发展多深的圆形横截面微内皮化的一个芯片的程序

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

根癌农杆菌介导的绿色微藻、寻常小球藻的基因工程

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