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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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>Agrobacterium tumefaciens</td>
			<td>Gold Biotechnologies</td>
			<td>Strain: AGL-1; Gift from Prof. Paul Hooykaas</td>
			<td>Genotype: C58 RecA (RifR/CarbR) pTiBo542DT-DNA</td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Enzo Life Sciences</td>
			<td>89151-400</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</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>Chlorella vulgaris</td>
			<td>University of Texas at Austin Culture Collection of Algae</td>
			<td>Strain: UTEX 395</td>
			<td>Wildtype strain</td>
		</tr>
		<tr>
			<td>CoCl2&#183;6H2O</td>
			<td>Sigma Aldrich</td>
			<td>C8661-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>CuSO4&#183;5H2O</td>
			<td>EMD Millipore</td>
			<td>CX2185-1</td>
			<td></td>
		</tr>
		<tr>
			<td>FeCl3&#183;6H2O</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>K2HPO4</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>KH2PO4</td>
			<td>VWR</td>
			<td>BDH9268-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H2O</td>
			<td>VWR</td>
			<td>97062-134</td>
			<td></td>
		</tr>
		<tr>
			<td>MnCl2&#183;4H2O</td>
			<td>JT Baker</td>
			<td>BAKR2540-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2CO3</td>
			<td>VWR</td>
			<td>BDH7971-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2EDTA&#183;2H2O</td>
			<td>JT Baker</td>
			<td>8993-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2MoO4&#183;2H2O</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>NaH2PO4 H2O</td>
			<td>Millipore Sigma</td>
			<td>CA80058-650</td>
			<td></td>
		</tr>
		<tr>
			<td>NaNO3&#160;</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>NH4Cl</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, KanR, pVS1, T-DNA(CaMV 35S/HygR/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&#160;</td>
			<td>SunBlaster</td>
			<td>210000000906</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy 4 Microplate UV/Vis spectrometer&#160;</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>ZnSO4&#183;7H2O</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。

Agrobacterium tumefaciens, a gram-negative soil-borne bacterium, possesses a unique interkingdom gene transfer ability, earning it the title &#34;natural genetic engineer&#34;1. This bacterium can transfer DNA (T-DNA) from a tumor-inducing plasmid (Ti-Plasmid) into host cells through a Type IV secretion system, resulting in the integration and expression of the T-DNA within the host genome1,2,3,4. In the natural setting, this process leads to tumor formation in plants, commonly known as crown gall disease. However, Agrobacterium can also transfer T-DNA into various other organisms, including yeast, fungi, algae, sea urchin embryos, and even human cells under laboratory conditions5,6,7,8.
Exploiting this natural system, Agrobacterium tumefaciens-mediated transformation (AMT) enables the random integration of gene(s) of interest into a host cell&#39;s nuclear genome by modifying the T-DNA region of the Ti-plasmid. For this purpose, a widely used AMT plant expression vector is pCAMBIA13029. Researchers can employ simple cloning workflows in E. coli before transferring the desired vector into A. tumefaciens for subsequent transfer to the host of interest.
Green microalgae are eukaryotes that share many similarities with land plants but are highly recalcitrant to genetic modification. However, genetic transformation plays a crucial role in both fundamental and biotechnological research of microalgae. In several microalgae species, particularly Chlamydomonas reinhardtii, genetic transformation via AMT has successfully introduced transgenes such as human interleukin-2 (hIL-2), the severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD), and two antimicrobial peptides (AMPs)10,11,12,13. Among these, Chlorella vulgaris, a less fastidious and fast-growing green algae species, holds significant potential for the sustainable production of carbohydrates, proteins, nutraceuticals, pigments, and other high-value compounds14. However, the lack of reliable tools for creating transgenic strains of C. vulgaris hampers its commercial progress. Since there have been only a limited number of published works utilizing AMT in C. vulgaris15, and given the considerable differences between plant and microalgae cultivation, optimizing the AMT protocol becomes essential.
In this study, researchers inserted green fluorescent protein (mGFP5) downstream of the Cauliflower Mosaic Virus (CamV) 35S promoter and added a histidine tag to use it as a reporter gene for protein expression. Transformants were selected using Hygromycin B, and after subculturing for over twenty generations, the transformation remained stable. The pCAMBIA1302 plasmid employed in this work can be readily adapted to contain any gene of interest. Furthermore, the method and materials presented can be adjusted for other green algae species with an active CamV35S promoter, as this promoter is used for Hygromycin selection.

作者聚焦：使用根癌农杆菌优化寻常小球藻的转化方案 

根癌农杆菌介导的绿色微藻、寻常小球藻的基因工程

The scope of the research focused on the development and optimization of a protocol for the transformation of chlorella vulgaris. We aimed to create a stable transgenic strains of chlorella vulgaris using agrobacterium tumefaciens and a specific plasmid, pCAMBIA1302, that allowed them to insert gene of interest and the potential for bio technological applications. Metabolic engineering and synthetic biology, microscopy and imaging, CRISPR-Cas9 gene editing, bioinformatics, computational modeling, remote sensing and satellite imaging, nanotechnology, bioprocessing, and downstream processing.This study introduces an optimized agrobacterium tumefaciens media transformation protocol for the microalgae chlorella vulgaris using a specific plasmid and marker. It lays groundwork for enhancing microalgae by technology, overcoming previous limitations in reliable transformation tools for C.vulgaris.

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

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。

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

Research

JoVE Journal

Bioengineering

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

2.2K 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 

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

This protocol outlines the utilization of Agrobacterium tumefaciens-mediated transformation (AMT) for integrating gene(s) of interest into the nuclear genome of the green microalgae Chlorella vulgaris, leading to the production of stable transformants.

科研

生物工程

Preparation of Electrocompetent Agrobacterium tumefaciens Culture for Microalgal Genetic Engineering

Preparation of Electrocompetent  Agrobacterium tumefaciens  Culture for Microalgal Genetic Engineering  

Start by diluting 0.5 microliters of the overnight cultures of Agrobacterium tumefaciens culture with 50 milliliters of autoclaved ultrapure water. Spread 10 microliters of this diluted culture on a lysogeny broth or LB plate. Incubate the plate at 28 to 30 degrees Celsius for one to three days.Now, inoculate a single colony in 20 milliliters of LB media supplemented with rifampicin. Incubate the culture overnight at 28 to 30 degrees Celsius. The next day, inoculate 500 milliliters of plain LB media in a shaker flask with nine milliliters of overnight culture.Place the cultures on a shaker at 28 to 30 degrees Celsius. Following incubation and dividing the cultures into tubes, centrifuge the chilled tubes at four degrees Celsius and 4, 000 G for 15 minutes. Using a pipette, remove the supernatant and resuspend each pellet in 50 milliliters of ice cold water.After centrifuging and discarding the supernatant, resuspend the pellet in 25 milliliters of ice cold water in each tube. Centrifuge as before, then resuspend the pellet in one milliliter of ice cold 10%glycerol. Now, combine the contents of all the tubes into a single 50-milliliter tube before centrifuging.Finally, after discarding the supernatant, resuspend the pellet in 400 microliters of ice cold glycerol.

用于微藻基因工程的电感受态 根癌农杆菌 培养物的制备

Electroporation of Agrobacterium tumefaciens for Genetic Engineering of Microalgal Cells

Electroporation of  Agrobacterium tumefaciens  for Genetic Engineering of Microalgal Cells  

Begin by adding 50 microliters of electrocompetent Agrobacterium tumefaciens cells into a pre-chilled 0.1-millimeter Q-vet. Add two microliters of pCAMBIA1302 to the Q-vet. now use an electroporator at 2, 400 volts to eelectroporate the cells.Immediately add one milliliter of LB media to the Q-vet and pipette the solution up and down to mix gently. Next, transfer the resuspended cells into a 1.5-milliliter microtube. Then incubate it at 24 to 30 degrees Celsius for a minimum of one hour to recover.Now plate the cells on antibiotic supplemented LB auger. Incubate the plates at 28 to 30 degrees Celsius for two to three days. Finally, inoculate a single colony in LB broth.Allow the culture to grow overnight. Then cryopreserve the plasmid-containing strain with a 50%glycerol solution at 80 degrees Celsius for future use.

根癌农杆菌的电穿孔用于微藻细胞的基因工程

Agrobacterium-Mediated Transformation of Chlorella vulgaris and Confirmation of Gene Integration via Colony PCR (cPCR)

Agrobacterium-Mediated Transformation of Chlorella vulgaris  and Confirmation of Gene Integration via Colony PCR (CPCR)  

Begin by pipetting out 0.5 milliliters of a log phase culture of chlorella vulgaris. Spread the culture on at Tris-Acetate-Phosphate or TAP Agar plate. Grow the culture for five days at 25 degrees Celsius.Next, inoculate 10 milliliters of supplemented LB broth with a loop full of electroporated agrobacterium tumefaciens culture in a shaker flask. Incubate the flask at 28 to 30 degrees Celsius and 250 RPM overnight. The next day, inoculate one milliliter of the overnight culture into 50 milliliters of supplemented LB.Incubate the flask at 28 to 30 degrees Celsius and 250 RPM.To co-cultivate the algal and bacterial cultures. First, transfer the agrobacterium culture into a 50 milliliter tube. Centrifuge the tube at 4, 000 G for 30 minutes at room temperature.Then, pipette out the supernatant to discard it and wash the cells twice using the induction medium. Next, add 25 milliliters of induction media onto the Chlorella Vulgaris culture plate. Transfer the cells into a 50 milliliter tube, then centrifuge at 4, 000 G for 15 minutes at room temperature.After disposing of the supernatant, combine the algal cell pellet with 200 microliters of the bacterial suspension. Shake the combined culture in a rotary incubator at 21 to 25 degrees Celsius, said at 150 RPM for one hour. Spread 200 microliters of the mixed culture onto induction medium plates, supplemented with 15 millimoles of glucose and incubate the plates in the dark at 21 to 25 degrees Celsius for three days.After three days, collect the microalgae into a flask using 10 milliliters of TAP media. Supplemented with 20 milligrams per liter of tetracycline. Incubate the flask in the dark for two days, maintaining a temperature between 21 to 25 degrees Celsius.Plate 500 microliters of the culture onto selective media supplemented with 20 milligrams per liter of tetracycline. Incubate the plates at 21 to 25 degrees Celsius in darkness for two days, before shifting them into an illuminated chamber. Select single colonies from the transformation plate and streak them onto TAP agar plates.To perform colony PCR, start by adding a small volume of the transformant algal cells to 10 microliters of sterile water. Boil the solution at 98 degrees Celsius for 15 minutes. Similarly process another PCR template for the confirmation of agrobacterium absence in the sample.Run the PCR samples on a DNA agarose gel with a ladder, to verify the size of the resulting fragments. Transformed colonies were able to grow on plates containing hygromycin with cefotaxime. The wild type colonies did not grow on the plates.Colonies resistant up to 70 milligrams per liter of cefotaxime were obtained. Colony PCR Amplicon of pCAMBIA1302 was absent in the algal samples. However MGFP5G in amplicon was detected in all three algal samples.Algal cultures that were repeatedly subcultures of Cefotaxime showed the absence of the virulence protein E2, indicative of plasmid absence. A significant difference in algal growth of the transformants and wild type strains were observed. Despite lower growth, the transformant had higher fluorescence levels when normalized for cell density.