Name: calcium chloride dna uptake in e. coli
Uploaded: 2023-04-30T07:10:31.000Z
Duration: 10 min 54 s
Channel: JoVE

K12 Standards

Florida Department of Education

High School

Life Science

SC.912.L.16

SC.912.L.16.7

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

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

本方案描述了利用氯化钙法的改良方法制备和转化感受态E. coli DH5α的操作步骤（12）。
1. 设置
<ol>
	<li>仪器设备
	<ul>
		<li>分光光度计</li>
		<li>Sorval 离心机（或同等设备）</li>
		<li>台式离心机</li>
		<li>加热块或水浴锅</li>
		<li>旋转摇床</li>
		<li>恒温培养箱</li>
		<li>凝胶制备板</li>
		<li>加样孔梳子</li>
		<li>电压源</li>
		<li>电泳槽</li>
		<li>紫外光源</li>
		<li>微波炉</li>
	</ul>
	</li>
	<li>溶液与试剂
	<ul>
		<li>LB 培养基（每 1000 mL H2O 中含 10 g 酪蛋白酶水解物、5 g 酵母提取物和 5 g 氯化钠）</li>
		<li>SOC 培养基（每升含 2% (w/v) 胰蛋白胨、0.5% (w/v) 酵母提取物、10 mM NaCl、2.5 mM KCl、10 mM MgCl2、10 mM MgSO4 和 20 mM 葡萄糖）</li>
		<li>CaCl2-MgCl2 溶液（80 mM MgCl2，20 mM CaCl2）</li>
		<li>1 M CaCl2 溶液（若立即进行转化），或含 10% (v/v) 甘油的 0.1 M CaCl2 溶液（若需将细胞冷冻保存以备后续使用）</li>
		<li>LB 琼脂平板</li>
		<li>LB 选择性琼脂平板（本实验中，由于所用质粒具有氨苄青霉素抗性，因此使用含氨苄青霉素 100 µg/mL 的 LB 琼脂平板）</li>
		<li>E. coli DH5α 菌株</li>
		<li>pUC19 质粒 DNA（100 pg/µL）</li>
		<li>QIAprep Spin Miniprep 试剂盒（Qiagen）</li>
		<li>HindIII 限制性内切酶</li>
		<li>1 kb plus DNA Marker</li>
		<li>低熔点琼脂糖</li>
		<li>1× TAE 缓冲液（40 mM Tris 碱，20 mM 乙酸，1 mM EDTA）</li>
		<li>溴化乙锭（10 mg/mL）</li>
	</ul>
	</li>
	<li>一般安全注意事项 
	E. coli DH5α 属于生物安全等级 1 级（BSL1）。该类微生物对健康成人几乎不构成感染风险。然而，操作时仍需谨慎处理该微生物。</li>
</ol>
重要提示：本方案中的所有步骤均需在无菌条件下操作，并在冰上或4°C环境中进行，除非另有说明。
2. 实验方案
<ol>
	<li>从保存在20%甘油中的大肠杆菌（E. coli）DH5α冻存菌种（LB培养基中过夜培养后制备）中，划线接种至LB琼脂平板上以分离单菌落。37°C培养过夜（16–20小时）。</li>
	<li>挑取单个菌落接种于试管中含3 mL LB液体培养基的培养管中。在37°C、210 rpm振荡条件下培养过夜（16–20小时）。</li>
	<li>测定过夜培养物的OD600值。用该过夜培养物接种100 mL LB液体培养基于1 L锥形瓶中，使初始OD600=0.01。在37°C剧烈振荡（210 rpm）培养，每15–20分钟用分光光度计监测OD600值，直至培养物达到OD600=0.35（约3小时）。 
	注意：为保证转化效率，细菌细胞必须处于对数生长中期。细胞密度最大应为108个细胞/mL，对于大多数大肠杆菌（E. coli）菌株，此值对应OD600=0.4。使用分光光度计测量OD600可准确判断细胞所处的生长阶段。若本方案用于其他细菌菌株，需通过校准实验确定特定OD600值对应的菌落形成单位（CFU）数量，以建立该相关性。</li>
	<li>将50 mL培养液转移至两个预冷的聚丙烯离心管中。将离心管置于冰上冷却20分钟。</li>
	<li>在4°C条件下，以2700g（Sorval GSA转子4100 rpm）离心10分钟，收集菌体。</li>
	<li>移除上清液。将离心管倒置于吸水纸或滤纸上，彻底去除残留液体。</li>
	<li>将每个菌体沉淀重悬于30 mL预冷的CaCl2-MgCl2溶液（80 mM MgCl2，20 mM CaCl2）中。先加入5 mL溶液，轻轻涡旋至沉淀完全溶解，再加入剩余25 mL溶液。</li>
	<li>重复步骤2.4。</li>
	<li>重复步骤2.5。</li>
	<li>若需立即进行转化，将每个菌体沉淀轻轻涡旋重悬于2 mL预冷的0.1 M CaCl2溶液中。若此方法无法完全重悬沉淀，可轻柔地用移液器上下吹打（避免产生气泡）。 
	或者，可将感受态细胞冷冻保存以备后续使用。制备感受态细胞冻存液时，将菌体沉淀重悬于含10%（v/v）甘油的2 mL 0.1 M CaCl2冰冻溶液中。该溶液必须保持低温。将细胞悬液分装至预冷的1.5 mL聚丙烯离心管中（每管160 µL）。立即置于干冰/乙醇浴中速冻，随后转移至-70°C冰箱保存。</li>
	<li>转化CaCl2处理的细胞时，取50 µL感受态细胞分别加入两个1.5 mL聚丙烯离心管中。向其中一管加入1 µL（100 pg）pUC19质粒DNA，另一管不加DNA作为阴性对照。轻柔混匀（避免产生气泡），冰上孵育30分钟。 
	注意：转化时所用DNA量不应超过50 ng，体积不超过10 µL。</li>
	<li>将离心管转移至预热的金属浴中，精确在42°C孵育45秒。 
	注意：热激是关键步骤，温度和时间均不得超出规定范围。</li>
	<li>立即转移至冰上，孵育2分钟。</li>
	<li>加入950 µL SOC培养基，37°C孵育1小时，使细菌恢复并表达质粒中编码的抗生素抗性标记。</li>
	<li>将10 µL细胞悬液加入1000 µL SOC培养基中（1/100稀释），另取100 µL细胞悬液加入1000 µL SOC培养基中（1/10稀释）。分别取100 µL稀释液及未稀释的对照样品接种至选择性平板上，用涂布棒均匀涂布。通常，接种1/100和1/10稀释度的样品各100 µL，可获得适量的菌落形成单位（cfu）。理想情况下，每板菌落数应在30–300之间，以便于计数且菌落彼此分离。实际cfu数量取决于转化效率（见数据分析与结果部分）。</li>
	<li>将平板置于37°C培养。转化成功的菌落应在12–16小时内出现（具体时间取决于菌株类型和筛选方法）。阴性对照平板上不应有菌落生长。</li>
	<li>计数转化所得的cfu/板（表1）。</li>
	<li>为验证转化子是否携带pUC19质粒，需进行质粒提取及酶切鉴定。为此，挑取单个菌落接种于含3 mL LB液体培养基的试管中。在37°C、210 rpm振荡条件下培养过夜（16–20小时）。</li>
	<li>按照试剂盒说明书，使用QIAprep Spin Miniprep试剂盒进行质粒小量提取。</li>
	<li>取1 µg纯化的pUC19质粒，用限制性内切酶HindIII在37°C酶切1小时。 
	注意：任何可在pUC19多克隆位点内切割的酶均可用于此步骤。</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>组分</td>
			<td>用量</td>
		</tr>
		<tr>
			<td>10X 限制性消化缓冲液</td>
			<td>2.5 µl</td>
		</tr>
		<tr>
			<td>质粒 pUC19</td>
			<td>1 µg</td>
		</tr>
		<tr>
			<td>HindIII</td>
			<td>1 µl</td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>20.5 µl（定容至 25 µl）</td>
		</tr>
	</tbody>
</table>
<ol start="21">
	<li>在含有1 µg/mL溴化乙锭的1%琼脂糖凝胶中，以95 V电压电泳1小时，同时上样分子量标记物、经酶切的pUC19 DNA以及等量未经酶切的pUC19 DNA。 
	注意：电泳时间与电压可能因所用设备不同而有所调整。</li>
	<li>在紫外光下观察凝胶，比较酶切与未酶切pUC19 DNA的大小（图2）（参见数据分析与结果部分）。 
	根据各个具体转化实验的目的，进行必要的步骤以验证转化结果。</li>
</ol>
<img alt="用于大小比较的电泳凝胶图，包含DNA标记物、未消化和HindIII消化的质粒。" class="xfigimg" src="/files/ftp_upload/10515/10515fig2.jpg" data-altupdatedat="1761830439000" data-alt="图2"> 
图2：从转化的DH5α细胞中回收的质粒DNA的酶切分析。 从转化的DH5α细胞中回收质粒DNA，用HindIII进行酶切，于1%琼脂糖凝胶中电泳，并使用紫外光源成像（步骤2.19至2.22）。
3. 数据分析与结果
为了计算转化效率（即细胞摄取外源DNA能力的指标），需要对转化后获得的菌落进行计数：
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>稀释度</td>
			<td>菌落形成单位（Cfu）</td>
		</tr>
		<tr>
			<td>1/100</td>
			<td>34</td>
		</tr>
		<tr>
			<td>1/10</td>
			<td>246</td>
		</tr>
	</tbody>
</table>
表1：转化实验中计数的菌落形成单位（cfu）。
转化效率（TE）是指将 1 µg 质粒转入一定体积的感受态细胞后所产生的菌落形成单位（cfu）的数量。多个参数会影响转化效率：质粒大小、细胞基因型、感受态制备时的生长阶段、转化方法等。在计算 TE 时，需考虑涂板前是否进行了稀释，并将该稀释倍数纳入总 cfu 数量的计算中。转化效率（TE）通过以下公式计算：
<img src="/files/ftp_upload/10515/10515eq1.jpg" alt="转染效率计算公式：TE=每个稀释度的菌落形成单位数（cfu）/(DNA 用量/μg)。" data-altupdatedat="1761830439000">
首先将菌落形成单位（cfu）除以DNA的微克数，在本例中为0.0001 µg。然后将结果除以稀释倍数。本例中采用了1/10的稀释度，并将1 mL溶液中的100 µL进行涂板（稀释倍数：1/10 × 100 µL / 1000 µL = 0.01）。
<img src="/files/ftp_upload/10515/10515eq2.jpg" alt="转化效率公式；显示CFU与DNA比例的方程，计算结果为2.46×10⁸ CFU/µg。" data-altupdatedat="1761830439000">

转化是一种将外源DNA导入细菌细胞的强有力方法，在实验室的许多分子生物学应用中具有关键作用。此外，转化在自然界中也发挥着重要作用，它使细菌细胞能够交换遗传物质，从而增加遗传多样性，并有助于在多种环境条件下获得不同的有益性状以利于生存。许多细菌菌株编码自然感受态所需的基因，但这些基因被诱导表达的具体条件仍不清楚，需要进一步研究来确定这些条件。

转化 e. coli 使用改良氯化钙法转化细胞

来源：Natalia Martin1，Andrew J. Van Alst1，Rhiannon M. LeVeque1 和 Victor J. DiRita1 
1 密歇根州立大学微生物学与分子遗传学系
细菌能够通过一种称为水平基因转移的过程交换遗传物质（脱氧核糖核酸，DNA）。整合外源DNA为细菌提供了一种机制，使其能够获得新的遗传性状，从而适应不断变化的环境条件，例如抗生素或抗体的存在（1），或自然生境中发现的分子（2）。水平基因转移有三种机制：转化、转导和接合（3）。本文将重点介绍转化，即细菌从环境中摄取游离DNA的能力。在实验室中，转化过程通常包括四个基本步骤：1）制备感受态细胞，2）将感受态细胞与DNA共孵育，3）细胞恢复培养，以及4）涂布细胞以培养转化子（图1）。
<img alt="细菌转化过程示意图，包含以下阶段：细胞准备、转化、恢复、涂布。" class="xfigimg" src="/files/ftp_upload/10515/10515fig1.jpg" data-altupdatedat="1761830439000" data-alt="图 1"> 
图 1：转化过程的总体步骤。 转化过程包括四个基本步骤：1）感受态细胞的制备，2）与 DNA 共孵育，3）细胞恢复，以及 4）涂布细胞以培养转化子。
要实现转化，受体细菌必须处于一种称为感受态的状态。某些细菌能够响应特定环境条件而自然进入感受态。然而，许多其他细菌无法自然进入感受态，或者该过程所需的条件尚不明确。将外源DNA导入细菌的能力在研究中具有多种应用：例如扩增特定的DNA分子、大量表达蛋白质、作为克隆实验的一个组成部分等。由于转化技术在分子生物学中的重要性，目前已发展出多种实验方案，用于在无法满足自然感受态条件的情况下人工诱导细胞进入感受态。人工感受态细胞的制备主要有两种方法：1）通过化学处理细胞；2）对细胞施加电脉冲（电穿孔）。前者根据具体实验方法使用不同的化学试剂，以促进DNA与细胞表面之间的相互作用；后者则利用电场在细菌细胞膜上形成孔道，使DNA分子得以进入。化学法诱导感受态最有效的方法是使用二价阳离子进行孵育，其中最常用的是钙离子（Ca2+）（4,5）。本文将描述由钙离子诱导产生感受态的实验流程（6）。该方法主要用于革兰氏阴性菌的转化，本实验方案也将以此为重点。
化学转化过程包含一系列步骤，即通过阳离子处理细胞以诱导其产生化学感受态。随后通过温度变化——热激——促进感受态细胞对外源DNA的摄取（7）。细菌细胞包膜带负电荷。在如大肠杆菌（Escherichia coli）等革兰氏阴性细菌中，外膜因含有脂多糖（LPS）而带负电（8）。这会导致同样带负电的DNA分子被排斥。在化学感受态诱导过程中，带正电的钙离子可中和这种电荷排斥作用，从而使DNA吸附到细胞表面（9）。钙离子处理及与DNA的孵育均在冰上进行。随后进行较高温度（42°C）的孵育，即热激处理。这种温度变化进一步促进DNA的摄入。细菌细胞需处于对数生长中期，才能耐受热激处理；在其他生长阶段，细菌对热过于敏感，会导致活力丧失，显著降低转化效率。
可用于转化的DNA来源有多种。通常，在大肠杆菌（E. coli）的大多数实验室操作中，使用的是质粒——一种小型环状双链DNA分子。为了使质粒在转化后能在细菌细胞中稳定维持，其必须包含复制起始位点（origin of replication），从而使质粒能够独立于细菌染色体进行复制。并非所有细菌细胞都能在转化过程中成功摄取质粒，因此转化后的产物是转化细胞与未转化细胞的混合物。为了区分这两类细胞群体，需采用筛选方法来识别已获得质粒的细胞。质粒通常携带可筛选标记（selectable markers），即编码某种有利于生长性状的基因（例如对抗生素或化学物质的抗性，或弥补生长所需营养缺陷的补偿功能）。转化后，将细菌细胞接种于选择性培养基上，只有成功转化的细胞才能生长。例如，若质粒赋予细胞对某种抗生素的抗性，则选择性培养基即为含有该抗生素的生长培养基。可通过多种不同方法进一步验证在选择性培养基上生长的菌落是否为真正的转化子（即已成功导入目标质粒的细胞）。例如，可使用质粒提取方法（10）从这些细胞中回收质粒，并通过酶切消化确认其大小；或者，也可采用菌落PCR方法来验证目标质粒的存在（11）。
本实验的目的是制备 E. coli DH5α 化学感受态细胞，采用氯化钙法的改良程序（12），并用质粒pUC19进行转化，以测定转化效率。 E. coli DH5α 菌株是分子生物学应用中常用的一种菌株。由于其基因型，特别是 recA1 和 endA1该菌株可提高插入片段的稳定性，并改善后续质粒DNA制备的质量。由于转化效率随DNA片段尺寸增大而降低，本实验方案选用尺寸较小的质粒pUC19（2686 bp）（载体图谱见https://www.mobitec.com/cms/products/bio/04_vector_sys/standard_cloning_vectors.html）。pUC19赋予氨苄青霉素抗性，因此选用氨苄青霉素作为筛选抗生素。

尽管转化效率受多种因素影响，但此类非商业化的感受态细胞制备方法通常每微克质粒可获得 106 至 107 个转化子。因此，本实验制备的感受态细胞转化效率达到 2.46 × 108 cfu/µg，远高于预期范围。当特定实验应用需要更高转化效率时，另有可用于制备超感受态细胞的补充方案（13）。
对转化细胞中回收的质粒DNA进行消化分析，结果表明该质粒的大小与预期的pUC19 DNA（2686 bp）一致。

转化 E. coli 使用改良氯化钙法转化细胞

来源：Natalia Martin1，Andrew J. Van Alst1，Rhiannon M. LeVeque1，和 Victor J. DiRita1  
1 密歇根大学微生物学与分子遗传学系，密歇根州...

细菌具有极强的适应能力，而实现这种适应的机制之一就是能够吸收外源DNA分子。细菌可以摄取的一种DNA类型称为质粒，它是一种环状DNA片段，通常携带一些有用的信息，例如抗生素抗性基因。细菌通过从外部来源整合新的遗传信息而发生遗传改变的过程被称为转化。利用大肠杆菌（Escherichia coli，或E. coli）可以在实验室中轻松完成转化实验。为了实现转化，大肠杆菌细胞必须首先处于感受态，即具备从环境中摄取DNA分子的能力。实现这一状态的实验方案出人意料地简单：将细胞在氯化钙溶液中短暂孵育。该孵育过程使细胞对DNA分子具有通透性。通过离心将细胞沉淀后，弃去上清液。随后将质粒DNA加入感受态细胞中。在细胞与DNA共同孵育后，混合物被短暂加热至42摄氏度，接着在冰上迅速冷却。这种热激处理促使DNA穿过细胞壁和细胞膜进入细胞内。然后将细胞在新鲜培养基中孵育。随后，将细菌置于37摄氏度环境下，使其修复细胞膜并表达抗性蛋白。那些摄入质粒的细胞将忠实地复制该DNA，并将其传递给后代，同时表达其中可能编码的任何蛋白质，包括介导抗生素抗性的蛋白。这些抗性基因可作为选择性标记，用于鉴定成功转化的细菌，因为未摄入质粒的细胞不会表达抗性基因产物。这意味着，当细胞被接种在含有相应抗生素的固体培养基上时，只有摄入了质粒的细胞才能生长。为进一步确认菌落中细胞的转化情况，可将这些细胞在液体培养基中过夜培养以增加产量，然后再从样品中提取DNA。一旦DNA被分离，即可进行诊断性的限制性内切酶消化。由于限制性内切酶可在DNA的特定位置进行可预测的切割，若成功转化了目标质粒，则酶切产物在凝胶电泳中应呈现出可预测的条带模式。例如，若将pUC19质粒用限制性内切酶HindIII切割，在凝胶上应观察到一条长度为2686个核苷酸的单一条带。在本实验中，您将使用 pUC19 质粒转化大肠杆菌 DH-5 Alpha 菌株，然后通过 DNA 琼脂糖凝胶电泳验证转化是否成功。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。随后，使用70%乙醇对工作区域进行消毒。现在，通过将一环细菌接种到无菌的LB琼脂平板上，并用新的接种环涂布细菌来制备化学感受态细胞。然后，在37摄氏度下将平板培养过夜。第二天，再次用70%乙醇对实验台进行消毒，并从培养箱中取出平板。用无菌接种环将单个分离良好的菌落接种到含有3毫升LB液体培养基的试管中。随后，在37摄氏度、210转/分钟振荡条件下过夜培养。次日，使用分光光度计测定过夜培养物的光密度。然后，向一个1升的锥形瓶中加入100毫升LB液体培养基，并以光密度为0.01的接种量接入过夜培养物。接着，将培养物置于37摄氏度振荡培养，每15至20分钟检测一次OD600，直至培养物进入对数生长中期。大约三小时后，将 50 毫升培养物转移至两个预冷的聚丙烯瓶中。然后将瓶子放回冰上冷却 20 分钟。接下来，通过离心收集细胞。弃去上清液，并将瓶子倒置在纸巾上。接着，用 5 毫升预冷的氯化钙-氯化镁溶液重悬细菌沉淀，轻轻涡旋直至沉淀完全溶解。然后，向溶解的细菌沉淀中再加入 25 毫升该溶液。按前述方法重悬另一份细菌沉淀。之后，重复离心操作，并弃去上清液。如果将直接进行转化，请通过轻轻旋转离心管，将每份感受态细胞沉淀重悬于2毫升预冷的0.1摩尔氯化钙溶液中。开始转化操作时，将50微升感受态细胞转移至两个已标记的1.5毫升聚丙烯离心管中。然后，向其中一根离心管加入1微升pUC19质粒DNA。轻轻混匀，避免产生气泡，并将两根离心管置于冰上孵育30分钟。孵育结束后，将离心管转移至预热的金属浴中，在42摄氏度孵育45秒。立即再次将离心管放回冰上，继续孵育2分钟。随后，向每根离心管中加入950微升SOC培养基，并在37摄氏度孵育1小时，使细菌恢复生长并表达质粒中编码的抗生素抗性标记。进行1比100稀释时，向一个1.5毫升离心管中加入990微升SOC培养基和10微升细胞悬液。然后进行1比10稀释，向另一个1.5毫升离心管中加入900微升SOC培养基和100微升细胞悬液。接下来，使用涂布棒将100微升稀释后的细胞悬液和100微升阴性对照分别涂布于含氨苄青霉素的选择性培养板上，并将培养板在37°C下孵育12至16小时。孵育结束后，计数每块平板上经转化获得的菌落形成单位（CFUs），并记录数据。为验证转化子是否含有pUC19质粒，用无菌接种环从平板上挑取单个、分离良好的菌落，接种至含有3毫升LB液体培养基的离心管中。随后，在37°C下振荡培养过夜。第二天，按照生产商说明书，使用质粒DNA小提试剂盒从3毫升培养物中提取质粒DNA。完成质粒提取后，取1微克纯化的pUC19质粒DNA，加入限制性内切酶，在37°C下酶切反应1小时。然后，在含1微克/毫升溴化乙锭的1%琼脂糖凝胶中，依次上样20微升分子量标记物、1微克酶切后的质粒DNA和1微克未酶切的质粒DNA。接着，在95伏电压下电泳运行1小时。最后，使用紫外成像仪观察凝胶结果。在本实验中，采用氯化钙法的改良方法制备大肠杆菌 DH5 Alpha 感受态细胞，随后用质粒 pUC19 进行转化，以测定转化效率。为计算转化效率，需使用记录的菌落形成单位（CFU）数值，例如 1:100 和 1:10 稀释度的数据，或其他 CFU 数在 30 至 300 之间的稀释度结果。首先，将记录的 CFU 数（本例中为 246）除以涂板时所用的 DNA 量（本例中为 0.0001 微克）。然后，将所得数值再除以稀释因子，从而得到以每微克 DNA 的 CFU 数表示的转化效率。本例中采用了 1:10 的稀释度，并将 1 毫升溶液中的 100 微升涂板，因此最终稀释因子为 0.01。在未消化质粒的泳道中，环状 DNA 可能呈现为两条或三条亮度不同的条带。这是因为未切割的环状 DNA 可以以多种构象状态存在，例如超螺旋、开环或更接近线性结构，而这些不同构象在凝胶中的迁移速率各不相同。对回收质粒 DNA 消化结果的分析表明，所用质粒的大小与 pUC19 DNA 的预期大小一致，为 2,686 个碱基对。

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使用自适应氯化钙程序对大肠杆菌细胞的转化

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. Transformation is the process in which bacteria take up free DNA from the environment. In the lab, it usually includes four steps: preparing competent cells, mixing cells with DNA, recovering the cells, and plating them so transformants can grow.

For transformation to occur, cells must be competent, which means they can take in DNA. Some bacteria become naturally competent under certain conditions. Others do not, or the conditions are not known. For that reason, scientists often make cells artificially competent. Two common approaches are chemical treatment and electroporation, which uses electric pulses to open pores in the cell membrane.

This protocol uses calcium ions, especially Ca2+, to create chemical competence. It is mainly used with Gram-negative bacteria. E. coli cells and their outer membrane carry negative charge, and DNA is also negatively charged. Calcium helps neutralize this repulsion so DNA can attach to the cell surface. The cells and DNA are kept on ice during this step, and then a heat shock at 42°C helps drive DNA into the cells.

The cells must be in mid-exponential growth phase before heat shock. At other growth stages, they are more sensitive to heat and may lose viability. That lowers transformation efficiency. The DNA used for transformation is usually a plasmid, which is a small circular double-stranded DNA molecule. To stay in the cell after transformation, the plasmid must have an origin of replication so it can copy itself independently of the bacterial chromosome.

Not every cell becomes transformed, so the result is a mix of transformed and non-transformed cells. To find the cells that received the plasmid, the procedure uses selection. Plasmids often carry selectable markers, such as antibiotic resistance genes or genes that rescue a growth need. After transformation, the cells are plated on selective media. In this case, the media contains ampicillin, which allows only the transformed cells to grow.

Calcium Chloride DNA Uptake in E. coli

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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.

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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。

See how calcium chloride makes E. coli competent for DNA uptake, then use heat shock and ampicillin selection to identify transformants.

Calcium Chloride DNA Uptake in E. coli | JoVE K12

Natalia Martin

Andrew J. Van Alst

Rhiannon M. LeVeque

Victor J. DiRita

Department of Microbiology and Molecular Genetics

Michigan State University

Concepts

Performing the Experiment

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 ...

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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 ...

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 ...

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

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.

Calcium Chloride DNA Uptake in E. coli

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