Name: using p1 phage to move antibiotic resistance in e. coli
Uploaded: 2023-04-30T07:10:46.000Z
Duration: 11 min 21 s
Channel: JoVE

K12 Standards

Florida Department of Education

High School

Life Science

SC.912.L.16

SC.912.L.16.7

phage-transduction-method-to-transfer-ampicillin-resistance-from

Phage Transduction: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

1. 设置
<ol>
	<li>在开始任何涉及微生物的操作之前，使用 70% 乙醇对工作区域进行消毒。始终穿戴必要的个人防护装备（实验服和手套）。</li>
	<li>确保 LB 琼脂平板（含 1× 氨苄青霉素）、市售 P1 噬菌体裂解液、氯仿、1 M 柠檬酸钠、甘油，以及一盒预先灭菌的塑料移液器吸头和涂布棒均放置在手边便于取用的位置。</li>
	<li>通过高压灭菌制备无菌 LB 培养基，并用其配制三份 1 mL 的 LB 盐溶液。
	<ol>
		<li> mM MgCl2（952.11–2.3803 μg）、5 mM CaCl2（11.098 mg）、0.1–0.2% 葡萄糖（100–200 μg）</li>
		<li> mM MgSO4（12.0366 mg）、5 mM CaCl2（11.098 mg）</li>
		<li> mM 柠檬酸钠（25.806 mg）</li>
	</ol>
	</li>
	<li>操作结束后，使用 70% 乙醇对所有表面及手套进行消毒，并洗手。</li>
</ol>
2. 实验方案
<ol>
	<li>供体噬菌体裂解液的制备
	<ol>
		<li>在含有1倍氨苄青霉素的LB培养基中，于37 °C、220 rpm振荡并通气条件下，过夜培养1 mL的供体W3110菌株大肠杆菌（E. coli）。</li>
		<li>将该过夜培养物按1:100稀释至1 mL新鲜LB培养基中，LB培养基中添加10–25 mM MgCl2、5 mM CaCl2和0.1–0.2%葡萄糖。</li>
		<li>在37 °C、220 rpm振荡并通气条件下，继续培养该细菌稀释液2小时。</li>
		<li>当细胞进入对数生长期初期（可见明显生长且轻微浑浊）时，加入40 μL市售P1噬菌体，并在37 °C、220 rpm振荡并通气条件下继续培养。
		<ol>
			<li>在加入噬菌体前，细胞在600 nm处测得的光密度值应约为0.4（6）。</li>
		</ol>
		</li>
		<li>监测培养物1–3小时，直至培养物发生裂解。
		<ol>
			<li>裂解将导致细胞碎片增加，同时浑浊度显著下降（即当培养液变得透明、可透光时，视为细胞已裂解）。</li>
		</ol>
		</li>
		<li>向裂解液中加入数滴（50–100 μL）氯仿，涡旋混匀。
		<ol>
			<li>氯仿可杀死残留的供体细胞，从而对噬菌体裂解液进行灭菌，仅保留噬菌体，并提高该裂解液的滴度。</li>
		</ol>
		</li>
		<li>以14,000 rpm离心2分钟，去除碎片，将上清液转移至新离心管中。</li>
		<li>加入数滴氯仿，于4 °C保存不超过一天。</li>
	</ol>
	</li>
	<li>转导
	<ol>
		<li>在LB培养基中，于37 °C、220 rpm振荡并通气条件下，过夜培养1 mL的受体J53菌株大肠杆菌（E. coli）。</li>
		<li>取100 μL供体噬菌体裂解液（2.1）加入1.5 mL微量离心管中，管盖打开，于37 °C孵育30分钟。
		<ol>
			<li>该孵育步骤可使P1裂解液中残留的氯仿充分挥发，避免其对受体细胞造成影响。</li>
		</ol>
		</li>
		<li>以6,000 rpm离心5分钟，轻柔沉淀受体菌细胞。</li>
		<li>将细胞重悬于300 μL含100 mM MgSO4和5 mM CaCl2的新鲜LB培养基中（P1噬菌体需要钙离子以维持感染性）。</li>
		<li>设置两个反应体系，使用受体细菌细胞和制备好的供体噬菌体裂解液：1）转导反应，混合100 μL受体J53菌株大肠杆菌（E. coli）与100 μL供体噬菌体裂解液；2）阴性对照，混合100 μL受体J53菌株大肠杆菌（E. coli）与100 μL含100 mM MgSO4和5 mM CaCl2的LB培养基。</li>
		<li>于37 °C、220 rpm振荡条件下孵育30分钟。</li>
		<li>加入1 mL LB培养基和200 μL 1 M柠檬酸钠（pH = 5.5），于37 °C、220 rpm振荡条件下孵育1小时。
		<ol>
			<li>柠檬酸钠通过螯合钙离子降低P1噬菌体的感染性，防止受体细菌被裂解。</li>
			<li>该孵育过程有助于氨苄青霉素抗性标记的表达。</li>
		</ol>
		</li>
		<li>以6,000 rpm离心5分钟，沉淀细胞。</li>
		<li>将细胞沉淀重悬于100 μL含100 mM柠檬酸钠（pH 5.5）的LB培养基中，涡旋混匀后，将两个反应的全部溶液分别涂布于两块LB琼脂平板上。
		<ol>
			<li>转导样品的LB平板应含1倍氨苄青霉素，阴性对照平板则不含氨苄青霉素。</li>
			<li>若平板上存在P1噬菌体污染，在制备冻存菌种前需重新划线纯化。</li>
			<li>若未去除噬菌体，则从这些菌落生长的培养物仅在钙离子螯合剂存在时才能生长。</li>
		</ol>
		</li>
		<li>从两块平板上各挑取约3–4个菌落，再次划线接种于两块已涂布100 μL 1 M柠檬酸钠（pH = 5.5）的LB琼脂平板上。
		<ol>
			<li>转导样品的LB平板应含1倍氨苄青霉素，阴性对照平板则不含氨苄青霉素。</li>
		</ol>
		</li>
		<li>于37 °C过夜培养，使无噬菌体污染的菌落生长。</li>
		<li>从两块平板上分别挑取菌落，接种于5 mL LB培养基中，于37 °C、220 rpm振荡并通气条件下过夜培养。</li>
		<li>使用DNA小提法从这些培养物中提取DNA，取总培养体积中的4.5 mL进行提取。
		<ol>
			<li>使用35 μL无核酸酶水洗脱DNA。</li>
			<li>使用Nanodrop测定所得DNA浓度。纯DNA的吸光度比值（A260/280）约为1.8。</li>
		</ol>
		</li>
		<li>取每份培养物剩余的0.5 mL，与等体积100%甘油混合，制备1 mL甘油菌种（1:1比例）。</li>
		<li>将细菌甘油菌种保存于-80 °C。</li>
	</ol>
	</li>
</ol>
3. 数据分析与结果
<ol>
	<li>通过qPCR确认转导
	<ol>
		<li>为六次qPCR反应配制两种qPCR主混合液，其中三次使用氨苄青霉素抗性基因的qPCR引物，另三次使用看家基因的qPCR引物（每反应14.5 μL）：12.5 μL qPCR缓冲液混合物 + 1 μL 正向引物 + 1 μL 反向引物。
		<ol>
			<li>本实验中使用了SYBR Green主混合液。</li>
			<li>看家基因引物设计用于扩增编码DNA旋转酶B的细菌基因内的DNA片段（7）。</li>
		</ol>
		</li>
		<li>对于每次qPCR反应，将每次反应中提取的100 μg DNA（10.5 μL）与14.5 μL qPCR主混合液混合。</li>
		<li>使用qPCR仪及表1所列的热循环程序，对六次反应中的氨苄青霉素抗性基因和看家基因进行扩增检测。</li>
		<li>根据qPCR生成的Cq值计算氨苄青霉素抗性基因的标准化转导效率（图3），以确认氨苄青霉素抗性基因的成功转导。
		<ol>
			<li>Cq值（即循环定量值）是指样品信号首次超过背景阈值时所对应的最早PCR循环数。较低的Cq值表示目标序列含量较高，反之亦然。</li>
			<li>可通过Cq值计算样品中目标基因的标准化转导效率，方法是用目标基因的Cq值减去看家基因的Cq值，得到ΔCq值，再通过公式2(-ΔCq)计算标准化转导效率。</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>变性</td>
			<td>时间</td>
		</tr>
		<tr>
			<td>变性</td>
			<td>94 °C</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td colspan="3">40 个循环：</td>
		</tr>
		<tr>
			<td>变性</td>
			<td>94 °C</td>
			<td>15 sec</td>
		</tr>
		<tr>
			<td>退火、延伸及荧光读取</td>
			<td>60 °C 或比最低引物 Tm 低 5 °C</td>
			<td>1 min</td>
		</tr>
	</tbody>
</table>
表1：qPCR热循环程序

通过噬菌体将基因转移至细菌或从细菌转移出来，虽然是一个自然过程，但在多种研究目的中已被证明极为有用。尽管存在其他基因转移方法，如转化和接合，但转导独特地利用了噬菌体；这不仅允许基因整合到宿主基因组中，还能将基因递送至对其他方法不敏感的多种细菌。这一过程在实验室中尤其有用，同时也被应用于近年来新兴的基因治疗领域，更具体地说，是在替代性基因治疗中。替代性基因治疗是一种利用细菌将治疗剂递送至靶组织的治疗策略，许多此类靶组织对其他递送方法不敏感，因而具有重要的临床意义（8,9）。

噬菌体转导：一种将氨苄青霉素抗性从供体转移至受体大肠杆菌的方法 e. coli

来源：Alexander S. Gold1，Tonya M. Colpitts1 
1 微生物学系，波士顿大学医学院，国家新发传染病实验室，马萨诸塞州波士顿
转导是一种通过噬菌体介导的细菌间基因交换形式，噬菌体（或称噬菌体）是一类专门感染原核生物的病毒。这种通过噬菌体将DNA从一个细菌转移到另一个细菌的方式，于1951年由Norton Zinder和Joshua Lederberg发现，他们将该过程命名为“转导”（1）。噬菌体最早于1915年由英国细菌学家Frederick Twort发现，随后在1917年又被法裔加拿大微生物学家Felix d'Herelle独立发现（2）。自那时起，人们对这些噬菌体的结构与功能进行了广泛表征（3），并将其分为两类。第一类为裂解性噬菌体，其在感染后于宿主细菌内大量复制，破坏细菌的代谢过程，裂解细胞并释放子代噬菌体（4）。由于这种抗细菌活性以及耐药菌日益增多，近年来裂解性噬菌体已被证明可作为抗生素的替代治疗手段。第二类为溶原性噬菌体，它们可通过裂解循环在宿主内复制，也可进入一种静止状态，将其基因组整合到宿主基因组中（图1），这一过程称为溶原化（lysogeny），且可在后续多代中被诱导产生噬菌体（4）。
<img alt="噬菌体生命周期示意图，DNA注入宿主细胞，病毒复制过程说明。" class="xfigimg" src="/files/ftp_upload/10517/10517fig1.jpg" data-altupdatedat="1761830439000" data-alt="图1"> 
图1：噬菌体对宿主细胞的感染。 噬菌体通过尾丝与受体（紫色）之间的相互作用吸附到细菌细胞壁上。一旦到达细胞表面，噬菌体便利用基板（黑色）不可逆地附着于细菌细胞，基板由可收缩的鞘（黄色）带动移向细胞壁。随后，噬菌体基因组（红色）进入细胞并整合到宿主细胞基因组中。
虽然细菌转导是一种自然发生的过程，但利用现代技术，该过程已在实验室环境中被改造用于将基因导入细菌。通过将目的基因插入溶原性噬菌体（如噬菌体）的基因组中，即可将这些基因转移至细菌基因组，并在这些细胞内实现表达。与其他基因转移方法（如转化法）使用质粒进行基因转移和表达不同，噬菌体基因组插入受体细菌基因组后，不仅有可能赋予该细菌新的性状，还可使自然发生的突变及细胞环境中的其他因素影响所转移基因的功能。
与其他水平基因转移方法（如接合）相比，转导对供体和受体细胞所需条件的要求相对灵活。只要供体和受体细菌菌株均对噬菌体具有感受性，即其细胞表面表达必要的噬菌体受体，那么任何能够容纳于所用噬菌体基因组内的遗传元件，均可从任意供体菌株转移至任意受体菌株。一旦该基因从供体基因组中被移出并包装进噬菌体颗粒，便可将其转移至受体细胞。转导完成后，必须筛选出含有目的基因的受体细菌。这可通过使用遗传标记实现，例如 FLAG 标签或聚组氨酸标签来标记目的基因，或利用基因本身的内在功能，如编码抗生素抗性的基因。此外，还可通过 PCR 进一步确认转导是否成功：使用针对目的基因内部区域的引物，并将扩增信号与阳性对照（含有目的基因的细菌）和阴性对照（经历与转导反应相同步骤但未加入噬菌体的细菌）进行比较。尽管细菌转导是分子生物学中一种有用的工具，但它在过去并持续在细菌进化过程中发挥着重要作用，尤其是在当前抗生素抗性日益增加的背景下。
在本实验中，利用细菌转导将编码对氨苄青霉素耐药性的基因从W3110菌株转移 E.coli 通过P1噬菌体将氨苄青霉素抗性基因转导至J53菌株（5）。本实验包含两个主要步骤：首先，从供体菌株制备含有氨苄青霉素抗性基因的P1噬菌体；其次，利用P1噬菌体介导的转导作用将该基因转移至受体菌株（图1）。实验完成后，可通过qPCR检测氨苄青霉素抗性基因是否成功转移（图2）。若转导成功，J53菌株将获得该抗性基因。 E. coli 将对氨苄西林具有抗性，且该抗性基因可通过qPCR检测。若实验失败，则无法检测到氨苄西林抗性基因，氨苄西林仍可作为有效抗生素作用于J53菌株。

<img alt="显示转导样本与阴性对照样本qPCR结果的转导效率柱状图。" class="xfigimg" src="/files/ftp_upload/10517/10517fig2.jpg" data-altupdatedat="1761830439000" data-alt="图 2"> 
图 2：通过qPCR验证转导成功。 通过比较转导反应与阴性对照反应中目的基因检测到的Cq值，并将这些值相对于一个看家基因进行归一化处理，可确认细菌转导已成功实现。

噬菌体转导：一种将氨苄青霉素抗性从供体转移至受体大肠杆菌的方法 E. coli

来源：Alexander S. Gold1，Tonya M. Colpitts1  
1 波士顿大学医学院微生物学系，国家新兴传染病实验室

细菌可以通过交换遗传物质快速适应快速变化的环境，其中一种方式是转导，即由细菌病毒介导的遗传物质交换。噬菌体（bacteriophage）常简称为phage，是一类感染细菌的病毒，其首先附着于宿主细胞表面，随后将其DNA注入细菌细胞内。接着，它会降解宿主细胞自身的DNA，并复制其病毒基因组，同时劫持细胞的合成机制以产生大量病毒蛋白。这些噬菌体蛋白随后会自我组装，并将噬菌体基因组包裹进衣壳，形成多个子代噬菌体。然而，由于DNA包装机制的保真度较低，偶尔噬菌体会将细菌DNA片段包装进噬菌体衣壳中。在诱导宿主细胞裂解后，子代噬菌体被释放；当此类噬菌体感染另一个宿主细胞时，便会将其前一个宿主的DNA片段转移进去。该DNA片段随后可能发生重组并永久整合到新宿主的染色体中，从而实现两个细菌之间的基因转移。在实验室中进行噬菌体转导需要以下要素：含有目的基因的供体菌株、缺乏该基因的受体菌株、能够感染这两种菌株的噬菌体，以及一种用于筛选转导细菌的方法。在大多数情况下，该方法为一种选择性固体培养基，它能够支持转导细菌的生长，同时抑制未转导细菌的生长。实验开始时，将含有目的基因的供体菌株接种于液体培养基中进行培养。当所有细菌均处于对数生长期并活跃分裂时，向培养物中接种目标噬菌体。经过三至四小时的孵育后，几乎所有细菌均已裂解并释放出噬菌体颗粒，此时将供体噬菌体裂解液接种至新培养的受体菌株培养物中。短暂孵育一小时后，培养物中应包含转导与未转导细菌细胞的混合物，通过将部分菌液涂布于适当的选择性固体培养基上来筛选转导细胞。经过进一步孵育，转导细胞应生长并增殖形成可见菌落。随后可挑选这些菌落，利用多种方法进一步验证转导是否成功，例如菌落PCR、DNA测序或定量PCR。开始实验操作前，应穿戴适当的个人防护装备，包括实验服和手套。接着，使用70%乙醇对工作区域进行消毒，并擦拭台面。此后，准备三份每份一毫升的 LB 盐溶液。现在，通过将 100 微升的大肠杆菌加入含有五毫升 LB 培养基并添加 500 微克氨苄青霉素的 15 毫升锥形管中，制备供体菌株培养物。然后，在 37 摄氏度下振荡培养过夜，通气并以 220 转/分钟振荡。第二天，从摇床培养箱中取出培养物前，先用 70% 乙醇擦拭工作台面。接着，通过将 10 微升供体菌液加入 990 微升添加了盐溶液的新鲜 LB 培养基中，将过夜培养物按 1:100 稀释。将细菌稀释液在37摄氏度、220 rpm振荡和通气条件下培养两小时。当细胞达到对数生长期初期时，从培养箱中取出培养物，向其中加入40微升P1噬菌体，再次孵育。继续监测细胞一至三小时，直至培养物发生裂解。随后，向裂解液中加入50至100微升氯仿，通过涡旋振荡混合。然后，离心裂解液以去除碎片，并将上清液转移至新的无菌管中。向上清液中加入几滴氯仿，于4摄氏度保存，保存时间不得超过一天。开始转导实验时，取1毫升受体菌株培养物。接着，将100微升供体噬菌体裂解液转移至1.5毫升微量离心管中，在37摄氏度下敞盖孵育30分钟，使残留的氯仿充分挥发。在供体噬菌体裂解液孵育的同时，通过轻柔离心收集受体菌株细胞。弃去上清液，将菌体沉淀重悬于300微升含有100毫摩尔硫酸镁和5毫摩尔氯化钙的新鲜LB培养基中。接下来，通过在微量离心管中混合100微升受体菌株和100微升供体噬菌体裂解液，建立转导反应体系。然后，通过混合100微升受体菌株和100微升含硫酸镁与氯化钙的LB培养基，设置阴性对照。孵育后，向两个离心管中各加入200微升1摩尔的柠檬酸钠和1毫升LB培养基，并通过轻柔吹打混匀。随后，当离心管孵育1小时后，通过离心轻轻沉淀细胞。离心后，弃去上清液，并将沉淀的细胞重悬于含有100毫摩尔柠檬酸钠的100微升LB培养基中。涡旋混匀溶液，然后将全部转导样品移至含1倍氨苄青霉素的LB琼脂平板上。最后，将阴性对照细胞混合物的全部体积移至不含氨苄青霉素的LB琼脂平板上。将平板在37摄氏度下过夜培养后，使用无菌移液器吸头从转导平板上挑取三至四个菌落，并划线接种到一个新的含有1倍氨苄青霉素和100微升1摩尔柠檬酸钠的LB琼脂平板上。对阴性对照样品，采用相同的涂板方法，接种至另一含有仅100微升1摩尔柠檬酸钠的LB琼脂平板上。随后，将平板在37摄氏度下过夜培养，以使不含噬菌体的菌落生长。第二天，用70%乙醇擦拭实验台面，然后从培养箱中取出培养板。使用无菌移液器吸头，从转导平板上挑取三个菌落，并将每个菌落分别加入含有5毫升LB培养基的独立离心管中。接着，从阴性对照平板上挑取三个菌落，加入另一管含有5毫升LB培养基的离心管中。将各培养物在37摄氏度下振荡培养过夜，振荡速度为220 rpm，并保证通气。按照之前演示的方法对实验台面进行消毒后，使用DNA小提试剂盒，根据制造商说明书从每种培养物中提取4.5毫升的DNA。然后，用35微升无核酸酶水洗脱DNA，并使用实验室分光光度计测定其浓度。最后，将两种细菌培养液剩余的0.5毫升分别与0.5毫升100%甘油混合，制备甘油保存菌种。为确认转导效果，首先配制两份用于24次qPCR反应的qPCR预混液。对于第一份预混液，在微量离心管中加入150 µL qPCR缓冲液混合物，并分别加入12 µL针对氨苄青霉素抗性基因设计的正向引物和反向引物。接着，通过在微量离心管中加入150 µL qPCR预混液，再分别加入12 µL针对看家基因设计的正向引物和反向引物，配制第二份qPCR预混液。每个qPCR反应中，将来自各反应的100微克实验DNA与14.5微升qPCR预混液混合。接下来，按照之前演示的方法准备其余反应。将反应体系转移至预热至94摄氏度的热循环仪中，然后启动程序。最后，利用qPCR生成的循环定量值（Cq值）计算氨苄青霉素抗性基因的标准化转导效率。将目的基因在各阴性对照和转导样本中的循环定量值（Cq值）进行列表统计。较低的Cq值，通常低于29个循环，如本示例中的转导样本所示，表明目标序列的含量较高。此处也列出了一个看家基因，用作上样对照，以标准化每个反应中的DNA量，并作为阳性对照以确保qPCR正常工作。只要上样量相同，该看家基因在每个样本中的检出率相对一致。接下来，为了计算每个样本的 ΔCq 值，需用每个样本的目标基因 Cq 值减去其看家基因的 Cq 值。例如，第一个阴性对照的 ΔCq 值为 13.54。然后，利用此处所示公式，根据该值计算每个样本的标准化转导效率。最后，可计算出每组样本的平均标准化转导效率。

Education

教育

JoVE Science Education

Advanced Biology

高级生物学

Microbiology

微生物学

噬菌体转导:将氨基青霉素耐药性从捐赠者转移到受体大肠杆菌的方法

using p1 phage to move antibiotic resistance in e. coli

Bacteriophage transduction moves DNA between bacteria by using a phage, or bacterial virus, as the carrier. In this process, a phage can deliver genetic material from one bacterium to another. The method is a form of horizontal gene transfer and is important in both bacterial evolution and laboratory genetics.

Phages were first described in the early 1900s, and transduction was named in 1951 by Norton Zinder and Joshua Lederberg. The transcript describes two main phage classes. Lytic phages multiply inside the host cell, disrupt metabolism, lyse the cell, and release new phage particles. Lysogenic phages can also enter a dormant state, where the phage genome integrates into the host genome and may be induced later.

Modern researchers can use this natural process to move genes into bacteria. A gene of interest can be placed in a lysogenic phage genome and then transferred into a recipient bacterium, where it can be expressed. This approach differs from transformation, which uses plasmids, and it can also be more flexible than conjugation because the donor and recipient only need to be permissive to the phage and have the right surface receptors.

After transduction, the recipient cells must be selected to find the ones that received the gene. Researchers can use a marker such as a FLAG-tag or polyhistidine-tag, or they can use the gene's own function, such as antibiotic resistance, for selection. PCR can also confirm the transfer by comparing the target signal with positive and negative controls.

The experiment described here used P1 bacteriophage to move the ampicillin resistance gene from the W3110 strain of E. coli to the J53 strain. The work had two main steps. First, P1 phage carrying the resistance gene was prepared from the donor strain. Second, the phage was used to transduce the gene into the recipient strain.

Successful transfer could then be checked by qPCR, or quantitative PCR. If the transduction worked, J53 would become resistant to ampicillin and the resistance gene would be detected by qPCR. If it did not work, the gene would not be detected, and ampicillin would still stop the J53 strain from growing.

Using P1 Phage to Move Antibiotic Resistance in E. coli

using-p1-phage-to-move-antibiotic-resistance-in-e-coli

creating a winogradsky column: a method to enrich the microbial species in a sediment sample

creating-winogradsky-column-method-to-enrich-microbial-species

Winogradsky Column Microbial Layers and Gradients

A Winogradsky column shows how microbial layers form in mud and water over time. It is a clear, enclosed ecosystem that helps scientists grow sediment microbes that are hard to culture in a test tube or on a petri dish. By keeping conditions close to a microbe’s natural habitat, the column makes it possible to study organisms that help drive Earth’s biogeochemical cycles. The column is built from pond or wetland sediment mixed with water in a transparent container. Extra sources of carbon,...

serial dilutions and plating: microbial enumeration

serial-dilutions-and-plating-microbial-enumeration

Counting Bacteria with Dilution Plates

Counting bacteria with dilution plates helps microbiologists estimate how many cells are in a sample. Because bacteria can grow quickly and may be hard to count directly, serial dilution and plating make the concentration easier to measure. These methods have been used for more than a century to study bacterial and viral loads in clinical, industrial, pharmaceutical, and academic labs. Serial dilution reduces a sample in steps. A small volume of the original solution is moved into a fixed...

enrichment cultures: culturing aerobic and anaerobic microbes on selective and differential medias

enrichment-cultures-culturing-aerobic-anaerobic-microbes-on-selective

Selective Media for Bacterial Isolation

Selective media help scientists isolate bacteria by supporting some microbes and blocking others. This lab also uses differential media, which reveal a visible chemical trait in the organism being grown. Together, these methods help identify bacteria from mixed samples. Prokaryotic cells live in almost every environment on Earth. They also show wide metabolic diversity, so the growth medium must supply the molecules they need for energy. Some microbes are extremophiles, meaning they live in...

pure cultures and streak plating: isolation of single bacterial colonies from a mixed sample

pure-cultures-streak-plating-isolation-single-bacterial-colonies-from

Streak Plate Method for Isolating Bacteria

The streak plate method is used to isolate single bacterial colonies from a mixed sample. It helps separate one bacterial type from another so a pure culture can be grown. This is important in both clinical and research settings, where bacteria often grow together in mixed cultures. Pure cultures are needed for later genetic and protein studies. They are also used to check whether a sample is pure and to identify infectious agents from clinical samples. Because different strains can affect...

16s rrna sequencing: a pcr-based technique to identify bacterial species

16s-rrna-sequencing-pcr-based-technique-to-identify-bacterial

Bacterial ID with 16S rRNA Gene Sequencing

Bacterial identification with 16S rRNA gene sequencing helps scientists tell bacterial species apart. It is used in microbial ecology to study biodiversity in environmental samples and in medical microbiology to help diagnose infected patients. Traditional methods include microscopy, growth on specific media, biochemical and serological tests, and antibiotic sensitivity assays. Molecular methods have changed how bacteria are identified. 16S ribosomal RNA (rRNA) gene sequencing is often faster...

growth curves: generating growth curves using colony forming units and optical density measurements

growth-curves-generating-growth-curves-using-colony-forming-units

Bacterial Growth Stages and Cell Counts

Bacterial growth stages and cell counts show how a culture changes over time. This page follows a batch culture as it moves through lag, exponential, stationary, and death phases. It also shows how colony forming units, or CFU, and optical density can be used to measure that growth. Lag phase is the time bacteria need to adjust to a new environment before fast division begins. During exponential phase, cells grow and divide at the fastest rate, and DNA replication, RNA transcription, and...

antibiotic susceptibility testing: epsilometer tests to determine mic values of two antibiotics and evaluate antibiotic synergy

antibiotic-susceptibility-testing-epsilometer-tests-to-determine-mic

Measuring Bacterial Resistance with E-Tests

Measuring bacterial resistance with E-tests helps show how strongly an antibiotic can stop growth. The method is used to find the minimal inhibitory concentration, or MIC, which is the lowest antibiotic level that prevents visible bacterial growth. It is a practical way to study resistance in bacteria and compare how different antibiotics perform. The E-test uses a plastic strip that contains a built-in antibiotic gradient. The strip is placed on an agar plate that has been inoculated with...

microscopy and staining: gram, capsule, and endospore staining

microscopy-and-staining-gram-capsule-and-endospore-staining

Bacterial Stains for Cell Walls and Spores

Bacterial stains help scientists see cell walls, capsules, and endospores with a compound light microscope. Many bacteria are hard to tell apart with light alone because they are small, colorless, and can look similar under the lens. Staining makes these hidden features visible and helps identify different bacteria. A compound microscope uses more than one lens to magnify a sample. Its objective lens collects light near the specimen, and the eyepiece enlarges the image. Many compound...

plaque assay: a method to determine viral titer as plaque forming units (pfu)

plaque-assay-method-to-determine-viral-titer-as-plaque-forming-units

Phage Plaque Test for Counting Viruses

Bacteriophages, or phages, are viruses that infect prokaryotic cells. The plaque assay is a classic way to count these viruses by measuring viral titer, which is the amount of virus in a sample. It was first described in 1952 and is still one of the most reliable methods for determining viral titer. Phages were identified in the early 20th century by Twort and d'Hérelle. They have since been recognized for their therapeutic value and for their role in human and global ecosystems. Interest in...

transformation of e. coli cells using an adapted calcium chloride procedure

transformation-e-coli-cells-using-an-adapted-calcium-chloride

Calcium Chloride DNA Uptake in E. coli

Calcium chloride treatment can help E. coli take up foreign DNA. This chemical transformation method is used in molecular biology to move a plasmid into bacterial cells and then identify the cells that received it. The protocol described here uses chemically competent E. coli DH5α cells and the plasmid pUC19 to measure transformation efficiency. Bacteria can exchange genetic material through horizontal gene transfer. The three main routes are transformation, transduction, and conjugation.

conjugation: a method to transfer ampicillin resistance from donor to recipient e. coli

conjugation-method-to-transfer-ampicillin-resistance-from-donor-to

Bacterial Gene Transfer by Conjugation in E. coli

Bacterial conjugation is a form of horizontal gene transfer in E. coli that moves DNA from one cell to another through direct contact. It was first discovered by Lederberg and Tatum in 1946. Unlike transformation or transduction, conjugation is a natural, one-way transfer of DNA from a donor cell to a recipient cell. This process can increase bacterial genetic diversity. It is often described as a type of bacterial mating. It has also helped drive the spread of antibiotic-resistant bacteria.

phage transduction: a method to transfer ampicillin resistance from donor to recipient e. coli

Bacteriophage transduction moves DNA between bacteria by using a phage, or bacterial virus, as the carrier. In this process, a phage can deliver genetic material from one bacterium to another. The method is a form of horizontal gene transfer and is important in both bacterial evolution and laboratory genetics. Phages were first described in the early 1900s, and transduction was named in 1951 by Norton Zinder and Joshua Lederberg. The transcript describes two main phage classes. Lytic phages...

microbiology

该集合展示了微生物调查的关键工具，包括适当的无菌技术和电镀，如何使用选择性培养基和丰富样品，以及混合或纯样品的培养方法。此外，它着眼于识别微生物分离物的常见方法，以及细菌基因操作的程序。

Learn how bacteriophage transduction uses P1 phage to move ampicillin resistance between E. coli strains and how qPCR confirms success.

Using P1 Phage to Move Antibiotic Resistance in E. coli | JoVE K12

Alexander Gold

Tonya M Colpitts

Department of Microbiology

Boston University School of Medicine & National Emerging Infections Diseases Laboratories

Concepts

Preparation of Donor Phage Lysate

Transduction

Data Analysis by Quantitative Polymerase Chain Reaction

Results

Genetic Lingo

Overview

An organism is diploid if it inherits two variants, or alleles, of each gene, one from each parent. These two alleles constitute the genotype ...

Punnett Squares

Overview

A Punnett square displays the possible genotypes offspring can inherit from two parental genotypes. If a trait&#x2019;s inheritance pattern ...

Monohybrid Crosses

Overview

In the 1850s and 1860s, Gregor Mendel investigated inheritance by performing monohybrid crosses in pea plants. He crossed two plants that were ...

Dihybrid Crosses

Overview

To determine whether traits are inherited together or separately, Gregor Mendel crossed pea plants that differed in two traits. These parental ...

Law of Segregation

When crossing pea plants, Mendel noticed that one of the parental traits would sometimes disappear in the first generation of offspring, called the F1 ...

Law of Independent Assortment

While Mendel&#x2019;s Law of Segregation states that the two alleles for one gene are separated into different gametes, a different question of how ...

Test Cross

Alleles are different forms of the same gene. Humans and other diploid organisms inherit two alleles of every gene, one from each parent.

An allele is ...

Pedigree Analysis

Overview

A pedigree is a diagram displaying a family&#x2019;s history of a trait. Analyzing pedigrees can reveal (1) whether a trait is dominant or ...

Use Mendel's laws of segregation and independent assortment to analyze patterns of inheritance.

SC.912.L.16.1

Trihybrid Crosses

Trihybrid Crosses
Some of Mendel&#8217;s crosses examined three pairs of contrasting characteristics. Such a cross is called a trihybrid cross. A ...

Chi-square Analysis

The chi-square test is a statistical hypothesis test. It is used to check whether there is a significant difference between an expected value and an ...

Multiple Allele Traits

Incomplete Dominance

Gregor Mendel&#39;s work (1822 - 1884) was primarily focused on pea plants. Through his initial experiments, he determined that every gene in a diploid ...

Lethal Alleles

Agouti: A Lethal Allele
Lucien Cu&#233;not discovered lethal alleles in 1905 while studying the inheritance of coat color in mice. The agouti gene is ...

Polygenic Traits

background-and-environment-affect-phenotype

Background and Environment Affect Phenotype

Although the genetic makeup of an organism plays a major role in determining the phenotype, there are also several environmental factors, such as ...

Discuss observed inheritance patterns caused by various modes of inheritance, including dominant, recessive, codominant, sex-linked, polygenic, and multiple alleles.

SC.912.L.16.2

Base-pairing and DNA Repair

The DNA Replication Fork

Lagging Strand Synthesis

The Replisome

Proofreading

Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template ...

Replication in Prokaryotes

DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the ...

Replication in Eukaryotes

In eukaryotic cells, DNA replication is highly conserved and tightly regulated. Multiple linear chromosomes must be duplicated with high fidelity before ...

Describe the basic process of DNA replication and how it relates to the transmission and conservation of the genetic information.

SC.912.L.16.3

The DNA Helix

Deoxyribonucleic acid, or DNA, is the genetic material responsible for passing traits from generation to generation in all organisms and most viruses. DNA ...

Mutations

Mutations are changes in the sequence of DNA. These changes can occur spontaneously or they can be induced by exposure to environmental factors. Mutations ...

Mutation, Gene Flow, and Genetic Drift

In a population that is not at Hardy-Weinberg equilibrium, the frequency of alleles changes over time. Therefore, any deviations from the five conditions ...

Explain how mutations in the DNA sequence may or may not result in phenotypic change. Explain how mutations in gametes may result in phenotypic changes in offspring.

SC.912.L.16.4

What is Gene Expression?

A gene is a stretch of DNA that serves as the blueprint for functional RNAs and proteins. Since DNA is comprised&#160; of nucleotides and proteins are ...

RNA Structure

The basic structure of RNA consists of a string of ribonucleotides attached by phosphodiester bonds. Although most RNA is single-stranded, it can form ...

Types of RNA

Three main types of RNA are involved in protein synthesis: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). These RNAs perform diverse ...

Transcription

Transcription is the synthesis of RNA from a DNA sequence by RNA polymerase. It is the first step in producing a protein from a gene sequence. ...

Bacterial RNA Polymerase

Eukaryotic RNA Polymerases

General Transcription Factors

Tissue-specific transcription factors contribute to diverse cellular functions in mammals. For example, the gene for beta globin, a major component of ...

RNA Polymerase II Accessory Proteins

Transcription Elongation Factors

pre-mrna-processing-modification-of-pre-mrna-ends

Pre-mRNA Processing: Modification of pre-mRNA Ends

In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or ...

Pre-mRNA Processing: RNA Splicing

Transfer RNA Synthesis

Ribosomal RNA Synthesis

The Nucleolus

Additional Subnuclear Structures

Translation

Translation is the process of synthesizing proteins from the genetic information carried by messenger RNA (mRNA). Following transcription, it constitutes ...

Ribosomes

Ribosomes translate genetic information encoded by messenger RNA (mRNA) into proteins. Both prokaryotic and eukaryotic cells have ribosomes. Cells that ...

tRNA Activation

Initiation of Translation

Termination of Translation

Improving Translational Accuracy

Nonsense-mediated mRNA Decay

Molecular Chaperones and Protein Folding

The Proteasome

Regulated Protein Degradation

Proteins: From Genes to Degradation

Explain the basic processes of transcription and translation, and how they result in the expression of genes.

SC.912.L.16.5

Chromatin Structure and RNA Splicing

Alternative RNA Splicing

Nuclear Export of mRNA

Discuss the mechanisms for regulation of gene expression in prokaryotes and eukaryotes at transcription and translation level.

SC.912.L.16.6

Molecular Cloning

Molecular cloning is a set of methods, which are used to insert recombinant DNA into a vector - a carrier of DNA molecules that will replicate recombinant ...

An Overview of Genetic Engineering

Genetic engineering &#8211; the process of purposefully altering an organism&#8217;s DNA &#8211; has been used to create powerful research tools and model ...

Recombineering and Gene Targeting

One of the most widely used tools in modern biology is molecular cloning with restriction enzymes, which create compatible ends between DNA fragments that ...

bacterial-transformation-the-heat-shock-method

Bacterial Transformation: The Heat Shock Method

Transformation is the process that occurs when a cell ingests foreign DNA from its surroundings. Transformation can occur in nature in certain types of ...

Bacterial Transformation: Electroporation

The term &#x201C;transformation&#x201D; refers cellular ingestion of foreign DNA.  In nature, transformation can occur in certain types of bacteria.  In ...

An Introduction to Transfection

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell ...

Transformation of E. coli Cells Using an Adapted Calcium Chloride Procedure

Source: Natalia Martin1, Andrew J. Van Alst1, Rhiannon M. LeVeque1, and Victor J. DiRita1
1 Department of Microbiology and Molecular Genetics, Michigan ...

Conjugation: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

Source: Alexander S. Gold1, Tonya M. Colpitts1
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases ...

CRISPR

Bacterial Transformation

Background

In early 20th century, pneumonia was accountable for a large portion of infectious disease deaths1. In order to develop an effective vaccine ...

Describe how viruses and bacteria transfer genetic material between cells and the role of this process in biotechnology.

A. DNA stores and transmits genetic information. Genes are sets of instructions encoded in the structure of DNA.

B. Genetic information is passed from generation to generation by DNA in all organisms and accounts for similarities in related individuals.

C. Manipulation of DNA in organisms has led to commercial production of biological molecules on a large scale and genetically modified organisms.

D. Reproduction is characteristic of living things and is essential for the survival of species.

Using P1 Phage to Move Antibiotic Resistance in E. coli

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