Name: light-controlled fermentations for microbial chemical and protein production
Uploaded: 2022-03-22T14:00:00
Description: Watch this Scientific Journal Video about Light-Controlled Fermentations for Microbial Chemical and Protein Production at JoVE.com

这项研究得到了美国能源部，科学办公室，生物和环境研究办公室奖编号DE-SC0019363，NSF CAREER奖CBET-1751840，皮尤慈善信托基金和Camille Dreyfus教师学者奖的支持。

1. 使用 酿酒酵母 光电光IVT7电路进行光控化学生产
<ol><li>应变结构<ol>
<li>获得具有 他的3 auxotrophy的菌株，因为该标记对于大多数现有的OptoINVRT质粒是必需的5。如果寻求 酿酒酵母原生基因的光遗传学调控，请构建一个菌株，其中删除该基因的任何内源性拷贝。</li>
		<li>对含有 OptoINVRT7 电路（如 EZ-L4395）的质粒进行线性化处理?...

<ol>
	<li>Figueroa, D., Rojas, V., Romero, A., Larrondo, L. F., Salinas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rise+and+shine+of+yeast+optogenetics.">The rise and shine of yeast optogenetics.</a> Yeast. 38 (2), 131-146 (2021).</li><li>Pouzet, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promise+of+optogenetics+for+bioproduction:+Dynamic+control+strategies+and+scale-up+instruments.">The promise of optogenetics for bioproduction: Dynamic control strategies and scale-up instruments.</a> Bioengineering. 7 (4), 151 (2020).</li><li>Venayak, N., Anesiadis, N., Cluett, W. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+characterization+of+rapid+optogenetic+circuits+for+dynamic+control+in+yeast+metabolic+engineering.">Design and characterization of rapid optogenetic circuits for dynamic control in yeast metabolic engineering.</a> ACS Synthetic Biology. 9 (12), 3254-3266 (2020).</li><li>Lalwani, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+control+of+the+lac+operon+for+bacterial+chemical+and+protein+production.">Optogenetic control of the lac operon for bacterial chemical and protein production.</a> Nature Chemical Biology. 17 (1), 71-79 (2021).</li><li>Zhao, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+regulation+of+engineered+cellular+metabolism+for+microbial+chemical+production.">Optogenetic regulation of engineered cellular metabolism for microbial chemical production.</a> Nature. 555 (7698), 683-687 (2018).</li><li>Baumschlager, A., Khammash, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biological+approaches+for+optogenetics+and+tools+for+transcriptional+light-control+in+bacteria.">Synthetic biological approaches for optogenetics and tools for transcriptional light-control in bacteria.</a> Advanced Biology. 5 (5), 2000256 (2021).</li><li>Dvorak, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exacerbation+of+substrate+toxicity+by+IPTG+in+Escherichia+coli+BL21(DE3)+carrying+a+synthetic+metabolic+pathway.">Exacerbation of substrate toxicity by IPTG in Escherichia coli BL21(DE3) carrying a synthetic metabolic pathway.</a> Microbial Cell Factories. 14, 201 (2015).</li><li>Hartline, C. 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长期以来，动态控制一直被应用于提高代谢工程和重组蛋白生产的产量4。酶表达的转变通常使用化学诱导剂（如IPTG21，半乳糖22和四环素23）来实现，但也使用温度和pH等工艺条件介导。基因表达的光遗传学控制消除了改变发酵参数或培养基组成的需要，使其成为传统诱导策略的易于应用的替代方案。打开或关闭灯光的容易程...

光遗传学是利用光响应蛋白控制生物过程的一种新策略，为动态控制微生物发酵以进行化学和蛋白质生产提供了一种新策略1，2。工程代谢途径的负担以及某些中间体和产品的毒性通常会损害细胞生长3。这种压力会导致生物质积累不良并降低生产力3。这一挑战可以通过将发酵暂时划分为生长和生产阶段来解决，这些阶段将代谢资源分别用于生物质积累或产品合成4。我们最近表明，在这种两相发酵中，从生长到生产的过渡可以通过照明条件的变化来诱导5，6，7。光输入的高可调性、可逆性和正交性8 为光控发酵提供了独特的优势，而这些发酵剂很难或不可能用用于传统两相发酵动态控制的化学诱导剂复制4，9，10，11。
来自乳酸红杆菌的蓝光响应性EL222蛋白已被用于开发几种光遗传学电路，用于酿酒酵母的代谢工程5，7，12，13。EL222包含一个光氧电压传感器（LOV）结构域，该结构域在蓝光激活（465nm）时经历构象位移，使其能够与其同源DNA序列（C120）13结合。将EL222融合到病毒VP16激活结构域（VP16-EL222）可产生蓝光响应转录因子，该转录因子可逆地激活酿酒酵母7和其他生物体14中合成启动子PC120中的基因表达。基于EL222的几个电路已经开发并用于酿酒酵母的化学生产，例如基本的光激活OptoEXP系统7，其中目的基因直接从PC120表达（图1A）。然而，在发酵生产阶段通常遇到的高细胞密度下的光穿透问题促使我们开发在黑暗中诱导的倒置电路，例如OptoINVRT和OptoQ-INVRT电路（图1B）5，7，13。这些系统分别利用酿酒酵母和克拉沙猪笼草的半乳糖（GAL）或奎宁（Q）调节剂，用VP16-EL222控制其相应的抑制因子（GAL80和QS），以抑制光照下的基因表达，并在黑暗中强烈诱导。结合OptoEXP和OptoINVRT电路，可以双向控制基因表达，实现两相发酵，其中生长阶段由蓝光诱导，生产阶段与黑暗（图2A）5，7。
在生产阶段使用光而不是黑暗来诱导基因表达将大大扩展光遗传学控制的能力，但也需要克服在这个发酵阶段通常遇到的高细胞密度的光穿透限制。为此，我们开发了称为OptoAMP和OptoQ-AMP的电路，可以放大对蓝光刺激的转录响应。这些电路分别使用VP16-EL222的野生型或超敏突变体来控制GAL或Q调节子的转录激活剂Gal4p或QF2的产生，从而在光照下实现增强的灵敏度和更强的基因表达12，13（图1C）。OptoAMP电路可以在光密度（在600nm下测量;OD600）值至少为40，仅约0.35%的照度（仅在约7%的体积表面上为5%的光剂量）。与OptoEXP相比，这显示出更高的灵敏度，OptoEXP需要接近100%的照明12。在高细胞密度下用光有效诱导基因表达的能力为发酵的动态控制开辟了新的机会。这包括在两个以上的时间阶段进行发酵，例如三相发酵，其中生长，诱导和生产阶段以独特的轻计划建立，以优化化学生产（图2B）12。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63269/63269fig1v2.jpg"> 图1：用于动态控制酿酒酵母的光遗传学电路。 OptoEXP、OptoINVRT 和 OptoAMP 电路基于光敏 VP16-EL222 系统。（A）在OptoEXP电路中，暴露于蓝光会导致VP16-EL222的构象变化和二聚化，从而暴露DNA结合结构域并允许从PC120转录。该图由Zhao等人修改为7。（B） OptoINVRT电路利用GAL（如图所示）或Q调节子在黑暗中诱导表达。在基于GAL的电路中，VP16-EL222和GAL4是本构表达的，而PC120驱动GAL80抑制器的表达（在基于Q的电路中，GAL4和GAL80分别被QF2和QS取代，并且使用合成的含QUAS的启动子代替GAL启动子）。在光照下，Gal80p阻止了PGAL1目的基因的激活。在黑暗中，GAL80不被表达，并通过将其融合到一个本构的degron结构域（小的棕色结构域）而迅速降解，这允许Gal4p激活PGAL1。该图由Zhao等人修改为5。（C） 光电放大器电路还使用 VP16-EL222 来控制 GAL（如图所示）或 Q 重稳态。在这些电路中，GAL80抑制器（或QS）被组成表达并融合到光敏的degron（小蓝色域），确保在黑暗中进行严格的抑制。PC120和超敏的VP16-EL222突变体控制表达的GAL4（或QF2）与光，其强烈激活光中的PGAL1（或含QUAS的启动子）。GAL衍生的电路可以使用工程形式的PGAL1，例如PGAL1-M或PGAL1-S，它们具有增加的活性，以及由GAL调节子（PGAL1，PGAL10，PGAL2，PGAL7）控制的野生型启动子。该图由 Zhao 等人修改而来。请<a href="https://www.jove.com/files/ftp_upload/63269/63269fig1v2large.jpg" target="_blank">点击此处查看此图的放大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63269/63269fig02.jpg"> 图 2：随时间推移的两相和三相发酵。 （A） 采用倒置回路操作的两相发酵包括光驱动的生长阶段和黑暗的生产阶段。在生长阶段，随着生产途径的抑制，生物质会积累。在达到所需的OD 600时，细胞被转移到黑暗中以代谢调整，然后重悬于新鲜培养基中以进行生产阶段。（B）在三阶段过程中，生长，孵化和生产阶段由独特的光照时间表定义，其中可能包括黑暗的生长期，脉冲孵育和完全照明的生产阶段。使用Biorender创建的图。 <a href="https://www.jove.com/files/ftp_upload/63269/63269fig02large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
还开发了光遗传学电路，用于动态控制大肠杆菌中的化学和蛋白质产生。OptoLAC 电路使用基于 YF1/FixJ 双组分系统6 的光响应 pDawn 电路控制细菌 LacI 抑制器（图 3）。与OptoINVRT5类似，OptoLAC电路旨在抑制光中的基因表达并在黑暗中诱导基因表达。使用 OptoLAC 电路的表达水平可以达到或超过标准异丙基β-d-1-硫代乳糖焦氰胺苷 （IPTG） 诱导达到的水平，从而保持化学诱导的强度，同时提供增强的可调性和可逆性6。因此，OptoLAC电路能够为大肠杆菌的代谢工程提供有效的光遗传学控制。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63269/63269fig03.jpg"> 图 3：用于动态控制 大肠杆菌的 OptoLAC 电路。 OptoLAC电路调整pDawn系统和lac操纵子，以实现黑暗中的激活和光照下的抑制。在黑暗中，YF1磷酸化FixJ，然后激活PFixK2 启动子以表达 cI 抑制剂。 cI 抑制因子阻止来自PR启动子的 lacI 抑制因子的表达，PR 启动子允许从含 lacO的启动子转录目的基因。相反，蓝光降低YF1网激酶活性，逆转FixJ磷酸化，从而逆转 cI 表达，从而抑制 lacI 的表达并阻止含 lacO的启动子的表达。该图已从Lalwani等人6修改而来。 <a href="https://www.jove.com/files/ftp_upload/63269/63269fig03large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
我们在这里描述了 酿酒酵母 和 大肠 杆菌用于化学或蛋白质生产的光控发酵的基本方案。对于酵母和细菌，我们首先关注由 OptoINVRT 和 OptoLAC 电路实现的光驱动生长阶段和黑暗诱导生产阶段的发酵。随后，我们描述了一种由OptoAMP电路实现的三相（生长，诱导，生产）光控发酵的协议。此外，我们还描述了如何将光遗传学控制的发酵从微孔板扩大到实验室规模的生物反应器。通过该协议，我们的目标是为化学或蛋白质生产进行光控发酵提供完整且易于重复的指南。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Light-controlled chemical production using S. cerevisiae</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well culture plate</td>
			<td>USA Scientific</td>
			<td>CC7672-7524</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar powder</td>
			<td>Thermo Fisher Scientific</td>
			<td>303991049</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Reynolds</td>
			<td>B004NG90YO</td>
			<td></td>
		</tr>
		<tr>
			<td>BioSpectrometer with &#956;cuvette</td>
			<td>Eppendorf</td>
			<td>6135000923</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue LED panel</td>
			<td>HQRP</td>
			<td>884667106091218</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-L439 OptoINVRT7 Plasmid</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>See Reference 1</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Thermo Fisher Scientific</td>
			<td>501879892 (G8270-5KG)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002403</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5510</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td>Yamato Scientific America</td>
			<td>SOU-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Celltreat</td>
			<td>229656</td>
			<td></td>
		</tr>
		<tr>
			<td>PmeI</td>
			<td>New England Biolabs</td>
			<td>R0560L</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum meter</td>
			<td>Apogee Instruments</td>
			<td>MQ-510</td>
			<td></td>
		</tr>
		<tr>
			<td>Replica-plating device</td>
			<td>Thomas Scientific</td>
			<td>F37848-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Replica-plating pads</td>
			<td>Sunrise Science Products</td>
			<td>3005-012</td>
			<td></td>
		</tr>
		<tr>
			<td>SC-His powder</td>
			<td>Sunrise Science Products</td>
			<td>1303-030</td>
			<td></td>
		</tr>
		<tr>
			<td>SC Complete powder</td>
			<td>Sunrise Science Products</td>
			<td>1459-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile sealing film</td>
			<td>Excel Scientific</td>
			<td>STR-SEAL-PLT</td>
			<td></td>
		</tr>
		<tr>
			<td>YPD agar plates</td>
			<td>VWR</td>
			<td>100217-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>Thermo Fisher Scientific</td>
			<td>R25005</td>
			<td></td>
		</tr>
		<tr>
			<td>Light-controlled protein production using E. coli</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6X SDS Sample Buffer</td>
			<td>Cepham Life Sciences</td>
			<td>10502</td>
			<td></td>
		</tr>
		<tr>
			<td>12% Acrylamide protein gels</td>
			<td>Thermo Fisher Scientific</td>
			<td>NP0341BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well culture plate</td>
			<td>USA Scientific</td>
			<td>CC7672-7524</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Reynolds</td>
			<td>B004NG90YO</td>
			<td></td>
		</tr>
		<tr>
			<td>BioSpectrometer with &#956;cuvette</td>
			<td>Eppendorf</td>
			<td>6135000923</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue LED panel</td>
			<td>HQRP</td>
			<td>884667106091218</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue G-250</td>
			<td>Thermo Fisher Scientific</td>
			<td>20279</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis cell</td>
			<td>Bio-Rad</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis power supply</td>
			<td>Bio-Rad</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth (Miller)</td>
			<td>Fisher Scientific</td>
			<td>BP97235</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002403</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5510</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Thomas Scientific</td>
			<td>SX0425-1</td>
			<td></td>
		</tr>
		<tr>
			<td>OptoLAC plasmids</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>See Reference 2</td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td>Yamato Scientific America</td>
			<td>SOU-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Celltreat</td>
			<td>229656</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum meter</td>
			<td>Apogee Instruments</td>
			<td>MQ-510</td>
			<td></td>
		</tr>
		<tr>
			<td>SOC medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>15544034</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>5382000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP1521</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-phase fermentation using S. cerevisiae</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Same materials as &#34;Light-controlled chemical production using S. cerevisiae&#34; protocol plus the following:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-L580 OptoAMP4 Plasmid</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>See Reference 10</td>
		</tr>
		<tr>
			<td>Chemical production in a light-controlled bioreactor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Reynolds</td>
			<td>B004NG90YO</td>
			<td></td>
		</tr>
		<tr>
			<td>Antifoam</td>
			<td>Sigma-Aldrich</td>
			<td>A8311</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioreactor with control station</td>
			<td>Eppendorf</td>
			<td>B120110001</td>
			<td></td>
		</tr>
		<tr>
			<td>BioSpectrometer with &#956;cuvette</td>
			<td>Eppendorf</td>
			<td>6135000923</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>VWR Scientific</td>
			<td>89501-620 (CS)</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue LED panel</td>
			<td>HQRP</td>
			<td>884667106091218</td>
			<td></td>
		</tr>
		<tr>
			<td>BPT tubing</td>
			<td>Fisher Scientific</td>
			<td>14-170-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Thermo Fisher Scientific</td>
			<td>501879892 (G8270-5KG)</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Fisher Scientific</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr>
			<td>M9 Minimal Salts</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1374401</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002403</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5510</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4OH Solution</td>
			<td>Sigma-Aldrich</td>
			<td>I0503-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td>Yamato Scientific America</td>
			<td>SOU-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum meter</td>
			<td>Apogee Instruments</td>
			<td>MQ-510</td>
			<td></td>
		</tr>
		<tr>
			<td>SC Complete powder</td>
			<td>Sunrise Science Products</td>
			<td>1459-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

light-controlled fermentations for microbial chemical and protein production

微生物细胞工厂为从可再生原料生产化学品和重组蛋白提供了一种可持续的替代方案。然而，通过基因修饰使微生物负担过重会降低宿主的适应性和生产力。这个问题可以通过使用动态控制来克服：酶和途径的诱导表达，通常使用基于化学或营养的添加剂，以平衡细胞生长和生产。光遗传学提供了一种非侵入性，高度可调和可逆的动态调节基因表达的方法。在这里，我们描述了如何建立工程 大肠杆菌 和 酿酒酵母 的光控发酵，以生产化学物质或重组蛋白。我们讨论了如何在选定的时间和剂量下应用光，以分离微生物生长和生产，以改善发酵控制和生产率，以及获得最佳结果的关键优化考虑因素。此外，我们还描述了如何为实验室规模的生物反应器实验实施光控制。这些方案有助于在工程微生物中采用光遗传学控制，以提高发酵性能。

微生物代谢的光遗传学调节已成功实施，以生产多种产品，包括生物燃料，散装化学品，蛋白质和天然产物5，6，7，12，13。这些过程中的大多数都是为了在光照下进行细胞生长而设计的（当低细胞密度对光穿透的挑战最小时），以及一旦细胞生长，黑暗就会诱导生产。使用?...

Watch this Scientific Journal Video about Light-Controlled Fermentations for Microbial Chemical and Protein Production at JoVE.com

Light-Controlled Fermentations for Microbial Chemical and Protein Production

微生物代谢的光遗传学控制为发酵过程提供了灵活的动态控制。这里的实验方案展示了如何建立蓝光调节的发酵，用于不同体积尺度的化学和蛋白质生产。

用于微生物化学和蛋白质生产的光控发酵

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

与典型的诱导剂相比，光遗传学已被用于实现几种化学物质和蛋白质的更高滴度。使用该协议，其他人可以通过基于光的控制来增强自己的过程。光遗传学控制是无创的，高度可调的和可逆的。这些品质允许简化微生物过程的优化，甚至开辟独特的机会，如三相发酵和多色度控制。对于任何新的化学品，最佳光照条件都会有所不同。因此，如果使用此协议中的参数观察到低产量，则应测试不同的光照条件。首先获得具有HIS3氧菌的酿酒酵母菌株，HIS3氧化菌是OptoINVRT质粒所必需的标志物。如果试图控制酿酒酵母的原生基因，请确保在继续之前删除该基因的内源性拷贝。要开始菌株构建，请对含有OptoINVRT7电路的质粒进行线性化，例如EZL439。如果使用EZL439，其中包含在光线下抑制PIGA一个启动子并在黑暗中激活它的组分，则在Pme1限制位点线性化。使用标准的乙酸锂转化方法，将质粒整合到HIS3营养菌株中。转化后，将细胞以150 G离心一分钟。将细胞轻轻重悬于200微升新鲜SC-HIS培养基中。将整个细胞体积平板接种到SC-HIS琼脂平板上，并在30摄氏度下孵育两到三天，直到出现菌落。使用标准醋酸锂转化方案制备感受态细胞。用含有您希望通过光遗传学方式控制 PIGA1M 或 PIGA1S 启动子下游的基因的质粒转化细胞。转化后，将培养物以150 G离心一分钟，并将细胞沉淀轻轻重悬于200微升新鲜SC滴出培养基中。将整个细胞体积接种在酵母提取物蛋白胨葡萄糖琼脂平板上，如果整合到Delta位点或SC滴出板中，如果与含有选择标记物的质粒转化。在恒定的蓝光下将细胞在30摄氏度下孵育16小时，以保持光遗传学控制的基因受到抑制。使用波长为465纳米的任何光源，并在板上方约40厘米处放置LED面板，使光强度约为每秒每平方米80至110微摩尔。使用量子计测量强度。如果整合到Delta位点，将板复制到酵母提取物蛋白胨葡萄糖板上，这些板含有一系列沸星浓度，每毫升400微克至每毫升1， 200微克，以选择各种整合拷贝数。将复制板在恒定或脉冲蓝光下在30摄氏度下孵育两到三天，直到菌落出现。要进行初步筛选，请从每个板中选择八个菌落，并用它们在24孔板的单个孔中接种一毫升补充有2%葡萄糖的SC-HIS培养基。在恒定的蓝光照射下，以每分钟200转的速度在30摄氏度下振荡过夜细胞。第二天，在新鲜的SC-HIS培养基中用2%葡萄糖稀释每种培养物，以获得600纳米处的光密度值范围为0.01至0.3，并在30摄氏度的恒定或脉冲光下以每分钟200转的振荡条件培养培养物，直到光密度达到2至9之间。然后，用铝箔包裹板，关闭灯板，并在30摄氏度下以每分钟200转的速度在黑暗中孵育板四个小时。将培养物在24孔板中以234 G离心5分钟，并将细胞重悬于一毫升含有2%葡萄糖的新鲜合成完全滴剂中。使用无菌微孔板密封胶带密封板以防止所需产品的蒸发。将密封的板在黑暗中以每分钟200转的速度在30摄氏度下发酵48小时。为了收获发酵，将板在234 G下离心五分钟，并将800微升上清液转移到1.5毫升微量离心管中。根据感兴趣的化学品，使用高效液相色谱法、气相色谱质谱法或其他分析方法分析，使用最适合所用仪器的样品制备技术进行分析。从每个板中选择八个菌落，并用它们在24孔板的单个孔中接种一毫升含有2%葡萄糖的SC-HIS培养基。在30摄氏度的黑暗中以每分钟200转的振动将细胞生长过夜。第二天早上，在新鲜的SC-HIS培养基中用2%葡萄糖稀释每种培养物至0.1光密度。用铝箔包裹板以防止暴露在光线下，并在30摄氏度的黑暗中以每分钟200转振荡生长培养物，直到它们达到3的光密度。然后，在30摄氏度下将板在脉冲光下孵育12小时，每分钟振荡200转。将培养物以234 G离心5分钟，然后重悬于含有2%葡萄糖的新鲜SC-HIS培养基中。使用无菌微孔板密封胶带密封板以防止所需产品的蒸发。将密封的板在光照下发酵48小时，以30摄氏度的速度以每分钟200转的速度振荡。在进行光遗传学发酵后，使用两个回路在诱导时以一系列细胞密度测试化学生产。与OptoINVRT1和2电路相比，OptoINVRT7电路显示出乳酸和异丁醇的高滴度，其诱导值的最佳细胞密度为7.0和8.75。使用OptoAMP电路的发酵分三个阶段进行研究，这些阶段由不同的轻型占空比定义。乳酸生产可以使用脉冲生长阶段和完全照明的感应和生产阶段进行优化。异丁醇生产采用脉冲生长阶段、全照射感应阶段和脉冲生产阶段进行优化。而柚皮素生物合成最好使用脉冲生长全照射诱导和黑暗生产阶段进行优化。OptoLAC系统已用于大肠杆菌中，以与使用IPDG化学诱导实现的滴度相当或高于转录因子FDER的滴度。OptoLAC回路已被应用于生产甲羟戊酸盐，并且产量超过使用IPDG感应实现的滴度。生物反应器水平的甲羟戊酸盐生产证明了光遗传学调节对微孔板水平以外的细菌中化学和蛋白质生产的可扩展性和可调性。重要的是要确保照明步骤的光强度在适当的范围内，并且对于需要黑暗的步骤，避免光污染。该技术为实现酵母中异丁醇的记录滴度，用光稳定联盟群体，甚至使用合成光控细胞器控制代谢通量铺平了道路。

Research

JoVE Journal

Bioengineering

Fluorescence detection methods for microfluidic droplet platforms

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

科研

生物工程

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

组合合成和高通量的蛋白质释放的聚合物薄膜和纳米粒子库

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

内含肽介导的人工蛋白水凝胶的合成

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

木质素下调<em&gt;玉米</em&gt;通过dsRNAi和Klason木质素分析

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

Production and Targeting of Monovalent Quantum Dots

生产和目标单价量子点

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

一种快速定量荧光方法蛋白靶向小分子药物筛选

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

一种新型生物反应器为不同的微生物群落的高密度栽培

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

利用自动 Microbioreactor 技术优化异种蛋白质生产的通用协议

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

大肠杆菌-表达自组装蛋白纳米颗粒的生产，用于需要三聚体异体的疫苗

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...