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Inexpensive and easy method for 6 fragment Golden Gate Assembly of a modular S/MARs mammalian expression vector and its variants
bioRxiv - Synthetic Biology Pub Date : 2022-03-08 , DOI: 10.1101/2021.09.04.458594
Adrian Ionut Pascu

Background A basic requirement for synthetic biology is the availability of efficient DNA assembly methods. Numerous methods have been previously reported to accomplish this task. One such method has been reported, which allows parallel assembly of multiple DNA fragments in a one-tube reaction, called Golden Gate Assembly. This study aims to further simplify that method and make it more suitable for small labs and students. Methods Prior to amplification of the parental plasmids used in building the modules were domesticated using a variation of SDM (Site Directed Mutagenesis) called SPRIP. After careful design and amplification of the desired modules, using a high fidelity polymerase, amplified PCR fragments that enter the one-step-one-pot reaction were stored in Zymo DNA/RNA Shield at -20 degrees C and thawed whenever needed to be used as fragments or modules in the assembly. The fragments were designed to posses unique overhangs using NEB Golden Gate assembly tool and Snapgene , amplification of modules was performed using a Q5 high fidelity polymerase from preexisting plasmids or gene fragments, clean-up of the PCR products (fragments) was performed in one tube per assembly using Zymo DNA Clean and Concentrator-5, assembled using BsaI and T4 ligase, DpnI digestion performed for eliminating the background plasmids that remain after the PCR reaction and the resulting assembled product was transformed into competent E.coli cells. Transformants were screened using diagnostic digest transformed in HEK293T cells and the fluorescence was evaluated using fluorescent microscopy and flow cytometry. Results Herein presented is a simple and inexpensive alternate protocol to build modular plasmids using the Golden Gate Assembly method. 7 S/MARs mammalian expression vectors were designed and constructed using 6 modules previously amplified by PCR and stored in the appropriate buffer to eliminate exo- and endonuclease activity and to protect the DNA from freeze thaw cycles. The existing modules were interchangeable and new modules were easily amplified and stored for use when needed. The mammalian expression vectors constructed showed the desired restriction pattern and GFP expression in bacteria and in mammalian cells. A comparison of 7 pNoname variants was conducted using flow cytometry. Interestingly, no pNoname variant harbouring the SV40 promoter showed expression in tested HEK293T cells. It appears that using the Ef1a promoter in combination with the BGH polyA signal provides the best expression in S/MARS vectors harboring the DTS40 region, as measured by flow cytometry. Conclusions Provided the design steps are respected and the fragments are stored and labeled appropriately, multiple plasmid variants and combinations of the pre-designed modules can be assembled in one day, easier and using less resources than the established protocols, with good efficiency. The simplicity of the design and the affordability of the method could make modular cloning of plasmid constructs more accessible to small labs and students.

中文翻译:

模块化 S/MARs 哺乳动物表达载体及其变体的 6 片段 Golden Gate 组装的廉价且简单的方法

背景合成生物学的一个基本要求是有效的 DNA 组装方法的可用性。先前已经报道了许多方法来完成这项任务。已经报道了一种这样的方法,它允许在单管反应中平行组装多个 DNA 片段,称为金门组装。本研究旨在进一步简化该方法,使其更适合小型实验室和学生。方法 在扩增用于构建模块的亲本质粒之前,使用称为 SPRIP 的 SDM(定点诱变)变体驯化。在仔细设计和放大所需模块后,使用高保真聚合酶,将进入一步一锅反应的扩增 PCR 片段储存在 -20 摄氏度的 Zymo DNA/RNA Shield 中,并在需要用作组装中的片段或模块时解冻。使用 NEB Golden Gate 组装工具和 Snapgene 将片段设计为具有独特的突出端,使用 Q5 高保真聚合酶从先前存在的质粒或基因片段中进行模块的扩增,在一个试管中进行 PCR 产物(片段)的清理每次使用 Zymo DNA Clean 和 Concentrator-5 组装,使用 BsaI 和 T4 连接酶组装,进行 DpnI 消化以消除 PCR 反应后残留的背景质粒,并将所得组装产物转化到感受态大肠杆菌细胞中。使用转化在 HEK293T 细胞中的诊断消化物筛选转化体,并使用荧光显微镜和流式细胞术评估荧光。此处提出的结果是一种简单且廉价的替代方案,用于使用金门组装方法构建模块化质粒。使用先前通过 PCR 扩增的 6 个模块设计和构建了 7 个 S/MARs 哺乳动物表达载体,并储存在适当的缓冲液中,以消除外切和内切核酸酶活性并保护 DNA 免受冻融循环的影响。现有模块是可互换的,新模块很容易放大和存储,以备需要时使用。构建的哺乳动物表达载体在细菌和哺乳动物细胞中显示出所需的限制模式和 GFP 表达。使用流式细胞仪对 7 个 pNoname 变体进行了比较。有趣的是,没有携带 SV40 启动子的 pNoname 变体在测试的 HEK293T 细胞中表达。通过流式细胞术测量,似乎使用 Ef1a 启动子与 BGH polyA 信号组合在含有 DTS40 区域的 S/MARS 载体中提供了最佳表达。结论 只要设计步骤得到尊重,片段被适当地储存和标记,多个质粒变体和预先设计的模块的组合可以在一天内组装,比既定的协议更容易,使用更少的资源,效率高。设计的简单性和方法的可负担性可以使小型实验室和学生更容易获得质粒构建体的模块化克隆。没有携带 SV40 启动子的 pNoname 变体在测试的 HEK293T 细胞中表达。通过流式细胞术测量,似乎使用 Ef1a 启动子与 BGH polyA 信号组合在含有 DTS40 区域的 S/MARS 载体中提供了最佳表达。结论 只要设计步骤得到尊重,片段被适当地储存和标记,多个质粒变体和预先设计的模块的组合可以在一天内组装,比既定的协议更容易,使用更少的资源,效率高。设计的简单性和方法的可负担性可以使小型实验室和学生更容易获得质粒构建体的模块化克隆。没有携带 SV40 启动子的 pNoname 变体在测试的 HEK293T 细胞中表达。通过流式细胞术测量,似乎使用 Ef1a 启动子与 BGH polyA 信号组合在含有 DTS40 区域的 S/MARS 载体中提供了最佳表达。结论 只要设计步骤得到尊重,片段被适当地储存和标记,多个质粒变体和预先设计的模块的组合可以在一天内组装,比既定的协议更容易,使用更少的资源,效率高。设计的简单性和方法的可负担性可以使小型实验室和学生更容易获得质粒构建体的模块化克隆。通过流式细胞术测量,似乎使用 Ef1a 启动子与 BGH polyA 信号组合在含有 DTS40 区域的 S/MARS 载体中提供了最佳表达。结论 只要设计步骤得到尊重,片段被适当地储存和标记,多个质粒变体和预先设计的模块的组合可以在一天内组装,比既定的协议更容易,使用更少的资源,效率高。设计的简单性和方法的可负担性可以使小型实验室和学生更容易获得质粒构建体的模块化克隆。通过流式细胞术测量,似乎使用 Ef1a 启动子与 BGH polyA 信号组合在含有 DTS40 区域的 S/MARS 载体中提供了最佳表达。结论 只要设计步骤得到尊重,片段被适当地储存和标记,多个质粒变体和预先设计的模块的组合可以在一天内组装,比既定的协议更容易,使用更少的资源,效率高。设计的简单性和方法的可负担性可以使小型实验室和学生更容易获得质粒构建体的模块化克隆。结论 只要设计步骤得到尊重,片段被适当地储存和标记,多个质粒变体和预先设计的模块的组合可以在一天内组装,比既定的协议更容易,使用更少的资源,效率高。设计的简单性和方法的可负担性可以使小型实验室和学生更容易获得质粒构建体的模块化克隆。结论 只要设计步骤得到尊重,片段被适当地储存和标记,多个质粒变体和预先设计的模块的组合可以在一天内组装,比既定的协议更容易,使用更少的资源,效率高。设计的简单性和方法的可负担性可以使小型实验室和学生更容易获得质粒构建体的模块化克隆。
更新日期:2022-03-08
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