Biology
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快速、经济、简单地生产无细菌细胞裂解物
Chapters
Summary October 29th, 2021
Please note that all translations are automatically generated.
Click here for the English version.
该协议描述了一种快速而简单的方法，使用工程化的 大肠杆菌 菌株生产细菌裂解物以实现无细胞基因表达，并且只需要标准的实验室设备。
Transcript
使用该协议，仅使用广泛使用的通用实验室设备即可生产高质量的细菌细胞裂解物，使大多数研究人员可以轻松获得无细胞基因表达。将程序细胞自溶与冻融或冻干循环相结合，可以创建一种实用，实用且具有成本效益的方法，用于快速生产用于无细胞基因表达的细菌裂解物。首先使用接种环将自溶大肠杆菌菌株的细胞划到含有每毫升50微克氨苄西林的LB琼脂平板上，然后将板在37摄氏度下孵育过夜。
第二天，使用移液器吸头，从琼脂平板中挑选单个菌落，并将其加入含有氨苄西林的LB培养基的培养管中。在37摄氏度下过夜种植这种发酵剂。第二天，将400微升的起始培养物加入一升锥形瓶中，该烧瓶含有400毫升2xYTPG培养基，每毫升补充50微克氨苄西林。
在37摄氏度下孵育培养物，以300 RPM振荡。使用分光光度计和具有1厘米路径长度的光学比色皿，定期测量培养物在600纳米处的光密度。当光密度超过1时，在测量前开始将培养物稀释五倍，以确保测量保持在典型实验室分光光度计的线性范围内。
一旦五倍稀释培养物的光密度达到0.3，从培养箱中取出烧瓶并继续制备裂解物。为了制备裂解物，通过在室温下以1， 800×g离心15分钟来收获细胞。倒出上清液，用移液管除去剩余的液体。
向沉淀中加入45毫升冷S30A缓冲液，并通过涡旋重悬沉淀。然后，称量空的50毫升离心管，然后将细胞转移到管中。再次离心细胞并丢弃上清液。
使用移液器吸出任何剩余的上清液。再次称量管子，并从显示的重量中减去空管的重量，以获得沉淀的重量。对于每一毫克细胞沉淀，加入两微升冷的S30A缓冲液，并补充两毫摩尔二硫甲状腺素。
然后，通过剧烈涡旋重悬细胞。接下来，通过将细胞置于负20或负80摄氏度的冰箱中冷冻细胞，直到沉淀完全冷冻，然后在室温水浴中解冻细胞并剧烈涡旋两到三分钟。将细胞在37摄氏度下孵育45分钟，以300RPM振荡，然后通过在透明离心管中以30，000×g在4摄氏度下离心45分钟来清除样品中的重细胞碎片。
用移液管小心地将上清液转移到新管中，注意不要干扰沉淀。如果转移的上清液被沉淀中的物质污染，则重复离心。将上清液转移到1.5毫升离心管中。
并以21， 000 x g或台式离心机的最大速度再次离心五分钟。将清除的自解物等分到所需的体积中，并在零下80摄氏度下储存直至使用。对于使用自溶物的无细胞基因表达，在冰上混合8微升自体溶物和8.9微升预混液。
将DNA加入8纳摩尔的终浓度，任何其他必要的试剂和水，以获得20微升的最终体积。将反应加入384孔微孔板中，并使用读板仪测量荧光时间过程和终点。使用具有稀释系列血浆DNA的自溶物进行GFP表达，即使使用一个纳摩尔DNA也能产生强烈的表达。
在比较市售裂解物和自溶物之间的GFP表达时，自溶物产生了相当水平的GFP。由不同研究人员在两个不同的实验室生产的裂解物批次之间的差异大约在两倍之内。三个最关键的变量是细胞沉淀与缓冲液的比例，转移无碎屑的上清液以及在最终反应中使用最佳的PEG和镁浓度。
一旦制备出提取物，就可以使用为无大肠杆菌无细胞表达系统开发的大量现有方案，例如ClpXP介导的降解或AHL介导的群体感应。
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