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Nov 2019

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Assembly of Genetic Circuits with the Mammalian ToolKit
用哺乳动物工具包组装遗传电路   

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Abstract

The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. The Mammalian ToolKit (MTK) is a Golden Gate-based cloning toolkit for fast, reproducible and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be assembled into transcriptional units and further weaved into complex circuits. These circuits are easily repurposed and introduced in mammalian cells by different methods.

Keywords: Cloning (克隆), Golden-gate (金门克隆), High-throughput (高通量), Toolkit (Toolkit), Library (文库), Mammalian (哺乳动物)

Background

Molecular cloning is a hallmark of modern biotechnology with the ability to repurpose recombinant DNA into a variety of genetic circuits that can represent a spectrum of purposes. However, a major limitation in exploring the permutations possible in construction of genetic circuits is the ability to rapidly prototype, test and implement improvements on circuit designs. For this to come to fruition, the time from designing genetic circuits to delivery into cells needs to be expedited from conventional cloning methods, such as Gibson cloning (Akama-Garren et al., 2016) or restriction digests. We designed a framework in which a conventional gene circuit is deconstructed into its constituent components such that one can easily swap these components to quickly assemble vast combinations, assessing how each iteration affects function. Inspired by earlier iterations of cloning toolkits (Weber et al., 2011a and 2011b; Duportet et al., 2014; Lee et al., 2015; Martella et al., 2017; Pérez-González et al., 2017; Halleran et al., 2018; Pollak et al., 2019), we adopted a Golden-gate cloning system where assembly of constituent parts into functional units can be accomplished in a one-pot fashion (Weber et al., 2011a and 2011b). To concretely realize the promise of this framework, we built and characterized over 400 DNA parts that encode a myriad of reagents. We named the curated library and cloning framework the Mammalian ToolKit (MTK) (Fonseca et al., 2019). With the MTK, swapping promoters, switching fluorophores, or rapidly prototyping protein tags constitutes changing one plasmid in a reaction. This is markedly different from having to redesign oligos and PCR verifying correct assembly for every use case, thus discouraging continued optimization of a construct of interest. The hierarchical nature of this system enables a library of parts to expand to a library of transcriptional units (TUs) that can be further combined to create multi-transcriptional unit vectors that are then delivered to cells. These TUs are delivery-agnostic, promoting their multiple uses. For example, the same TUs used in a circuit delivered to a specific locus via CRISPR-Cas9 homologous recombination can be recycled in a PiggyBac transposase transfection to be randomly integrated. While the initial time from part verification to delivery into cells is comparable to conventional cloning (approximately 4 days), the repurposing of parts and TUs allows vast combinations of circuits to be assembled in only 2 days.

Despite the tremendous advantages the MTK presents, the barrier to entry may still dissuade those without a cloning background. To ameliorate the activation energy for adoption across all disciplines, here we present instructions on how to utilize the current library to assemble novel circuits, and a step-by-step protocol to “domesticate” new parts (that is to make them compatible with Golden Gate-based assembly of the MTK), assemble Transcriptional Units (TU), and create varied multi-transcriptional units (multi-TU). It is our goal to make this toolkit widely accessible and available to enhance the development of mammalian expression systems towards new and exciting discoveries in biology.

Materials and Reagents

  1. Sterile tips for micropipettes (Green-PakTM SpaceSaverTM stacked refills for Rainin pipettes)
  2. PCR microtubes (BioExpress, catalog number: T-3135-2 )
  3. Sequencing primer MTK_P072 (gagcctatggaaaaacgc)
  4. Sequencing primer MTK_P073 (gggcgtaatttgatatcg)
  5. LB Broth (Sigma-Aldrich, catalog number: L3022 )
  6. LB Broth with agar (Lennox) (Sigma-Aldrich, catalog number: L2897 )
  7. Chloramphenicol (Spectrum Chemical, catalog number: 40310016-2 ) (working concentration: 100 mg/ml)
  8. Carbenicillin solution (Spectrum Chemical, catalog number: 40310030-2 ) (working concentration: 25 mg/ml)
  9. Kanamycin sulfate (Sigma-Aldrich, catalog number: k4000 ) (working concentration: 100 mg/ml)
  10. T4 DNA Ligase (NEB, catalog number: M0202L )
  11. Poly(ethylene glycol) (PEG) (Millipore-Sigma, catalog number: 81268 )
  12. T4 Polynucleotide Kinase (NEB, catalog number: M0201L )
  13. T4 DNA Ligase Reaction Buffer (10x) (NEB, catalog number: B0202S )
  14. BsaI-HFv2 (NEB, catalog number: R3733S )
  15. FastDigest Esp3I (Thermo Fisher Scientific, catalog number: FD0454 )
  16. Miniprep kit (Thermo Fisher Scientific, catalog number: FERK0503PR )
  17. T4 DNA ligase + 2.5% PEG (see Recipes)

Equipment

  1. Micropipettes (Rainin Pipet-Lite XLS)
  2. Incubator shaker (New BrunswickTM Innova® 44, catalog number: M1282-0000 )
  3. Gel Documentation System (Protein Simple AlphaImager HP)
  4. Thermocycler (Bio-Rad C1000, catalog number: 1851148 )
  5. NanoDrop (Thermo ScientificTM NanoDropTM 2000c, catalog number: ND2000 )

Procedure

  1. Overview
    The process that generates genetic circuits using the MTK is shown in Figure 1. Parts 1-8 of the MTK library can be ordered from Addgene (https://www.addgene.org/browse/article/28197510/) or generated from oligos and PCR fragments. Domestication of new parts is done by a BsmBI Golden Gate assembly into a chloramphenicol resistant destination vector and by selection of white colonies. From the library of parts, transcriptional units (TUs) can be built by assembling Parts 1-8 with a BsaI Golden Gate reaction and selection of white, carbenicillin resistant clones. TUs, can be delivered as is or further combined, alone or together with additional TUs, into a Kanamycin resistant destination vector. This assembly is done with a BsmBI Golden Gate reaction which generates plasmids ready for introduction into mammalian cells.


    Figure 1. Overview of MTK cloning procedure. A. Parts are cloned from Oligos or PCR products into a Chloramphenicol-resistant domestication vector using a BsmBI Golden Gate assembly. Parts, either newly cloned or from the library available at Addgene are combined to produce Carbenicillin-resistant Transcriptional Units (TU). For delivery into mammalian cells, single or multiple TUs can be further combined to produce Kanamycin-resistant Multiple Transcription Units (Multi-TU). B. Standard TUs are composed of 8 parts. Part 1 contains the 5’ connector, Part 2 is the 5’UTR (typically a promotor), Part 3 includes the coding sequence, Part 4 is the 3’UTR (typically a terminator), and Part 5 includes the 3’ connector. Parts 6 through 8 are not shown and usually encode the bacterial resistance marker.

  2. Part domestication
    1. Existing Parts
      1. Order part from Addgene (https://www.addgene.org/browse/article/28197510/).
      2. Streak bacteria in LB agar plate with corresponding antibiotic.
      3. Pick a single white colony with a sterile microtip and inoculate 5 ml LB media with corresponding antibiotic.
      4. Grow bacteria for 16 h at 37 °C at 250 rpm in a shaker.
      5. Prepare plasmid DNA using Thermo Fisher Miniprep kit, following the manufacturer’s instructions.
      6. Measure the concentration of DNA on a NanoDrop.
      7. Sequence plasmid with primers MTK_P072 and MTK_P073 to assure presence of the correct part.
    2. PCR
      1. Forward primers are designed as (N4)CGTCTCNTCGGTCTCN(X4-6)(Y16-22).
        N is any base.
        X are part type-specific BsaI overhang bases. See Table 1.
        Y are bases homologous to the specific part and that allow PCR.
      2. Reverse primers are designed as (N4)CGTCTCNGGTCTCN(X4-6)(Y16-22).
        N is any base.
        X are part-specific BsaI overhang bases. See Table 1.
        Y are reverse complement bases homologous to the specific part and that allow PCR.
      3. Primers are ordered as custom 25 nmole DNA Oligos from idtDNA (https://www.idtdna.com/)
      4. For Part 1 (Connectors), and Part 5’ (Reverse connectors), an additional overhang needs to be added to the forward primer (see Table 2).
      5. For Part 5 (Connectors), and Part 1’ (Reverse connectors), an additional overhang needs to be added to the reverse primer (see Table 2).
      6. For Parts 2, 3, 4 that encode sgRNAs for spCAS9, the following oligos are ordered as custom 25 nmole DNA Oligos from idtDNA:
        TGTTTG(N20)G
        TAAAC(N’20)CA
        N is the guide-specific sequence and N’ its reverse complement

        Table 1. Primers for Part domestication


        Table 2. Connector-specific overhangs


    3. Removal of internal BsmBI and BsaI sites
      1. If internal BsaI or BsmBI sites exist, additional primers that flank these sites are required. These will create additional PCR products that are pooled together in a Golden Gate reaction to generate an MTK-compatible part. The following requirements need to be followed:
        1. Primers should use BsmBI sites in forward direction to ensure they are not incorporated in the final product.
        2. Overhangs generated by PCRs should be as different as possible from the overhangs generated for part building (TCGG and GGCT). For example CATA and not TAGG.
        3. Point mutations to remove BsaI/BsmBI sites should be silent in coding DNA sequences.
        4. Point mutations to remove BsaI/BsmBI sites should maintain CG content in non-coding DNA sequences.
      2. Example of primers used for removal of internal BsaI site in domestication of ERKKTR (ERK Kinase Translocation Reporter, MTK3_021, Figure 2). Overhang in the example is AGGA.


        Figure 2. Part of ERKKTR sequence before domestication. Bsal site is highlighted in yellow. Both forward and reverse primers encode BsmBI sites for posterior Golden Gate assembly. Additionally, reverse primer has A to T substitution that maintains G48, but removes Bsal from the final product.

      3. To facilitate part domestication for coding sequences, we have created a python script (available here: https://github.com/weinerlab/mtk_primer_generator) that generates candidate primers given an input sequence and a part number. The script finds BsmBI and BsaI restriction sites that need to be removed and then generates a list of potential silent mutations that are capable of removing each restriction site. Finally, it checks combinations of these potential mutations for general compatibility with Golden Gate assembly and for mutual compatibility with one another, enabling a streamlined, one-pot Golden Gate assembly of parts.
    4. Golden-gate reaction for PCR-generated parts
      1. PCR generated (He, 2011) or gene block encoded parts are domesticated in a PCR microtube, using the reaction mixture shown in Table 3.
      2. Run the protocol described in Table 4 in a thermocycler.

        Table 3. Golden Gate reaction setup for Part domestication


        Table 4. Thermocycling conditions for Golden Gate reaction


      3. If internal BsmbI sites must be retained in the domesticated part, the third step of the thermocycling reaction (Table 4, 37 °C, 10 min) is removed from the protocol.
      4. Transform bacteria as described in Sharma et al. (2017).
      5. Proceed from Step B3 in domestication of existing parts.
        Usually, pick 2 white colonies (see Notes) in case the PCR has added a mutation in the newly cloned part.
    5. Golden-gate reaction for oligo-generated parts
      1. Oligos for oligo-generated parts are first phosphorylated by incubating the following mixture at 37 °C for 1 h in a PCR microtube (Table 5).

        Table 5. Oligo Annealing reaction setup


      2. Dilute the phosphorylated oligos with 190 μl of distilled water.
      3. To anneal the oligos, a thermocycler protocol was prepared to hold the temperature of the reaction at 96 °C for 6 min, and ramp down 0.1 °C per second to 23 °C. The reaction is then held at 23 °C indefinitely.
      4. Domestication is performed using the reaction mixture described in Table 3, replacing the PCR fragments with the same volume of annealed oligos. For spCAS9 sgRNA domestication, MTK0_027 is replaced by MTK0_001.
      5. Proceed from Steps 4c to 4e as described above.

  3. Transcriptional Unit (TU) assembly
    1. Single TU
      1. Transcriptional units (TUs) are defined by Parts 1 through 8 from the MTK library. If the desired circuit requires only one TU, we recommend assembly of the TU with MTK678_001, and the appropriate Part 1 LS connector and Part 5 RE connector. This will allow the recycling of the TU for different methods of delivery into mammalian cells.
      2. Single TUs are built using the reaction mixture described in Table 6.
        If additional parts are required (e.g., instead of Part 3, using Part 3a and Part 3b), change volume of distilled water so reaction volume is in total equal to 10 μl.

        Table 6. Golden Gate reaction setup for TU assembly


      3. Proceed with Golden Gate reaction as described in Table 4.
      4. Transform bacteria as described in Sharma et al. (2017).
      5. Pick two single white colonies (see Notes) with a sterile microtip and inoculate 5 ml LB media with Carbenicillin.
      6. Grow bacteria for 16 h at 37 °C at 250 rpm in a shaker.
      7. Prepare plasmid DNA using Thermo Fisher Miniprep kit, following the manufacturer’s instructions.
      8. Measure the concentration of DNA on a NanoDrop.
      9. Digest plasmids with NotI or other appropriate restriction enzymes, following the manufacturer’s instructions, and verify correct size of fragments.
      10. Example of TU encoding the CAG-driven expression of ERKKTR fused to the fluorescent protein mAzamiGreen, with the bgh polyadenylation signal (Figure 3).


        Figure 3. Assembly and verification of a single TU encoding ERKKTR. A. Plasmids required for the assembly of TU for the expression of mAzamiGreen-fused ERKKTR. Plasmids enconding connectors LS and RE (highlighted in red) are essential for the generation of single TU circuits. B. Assembled ERKKTR encoding TU. Different parts used in assembly are shown in different colors and Notl sites. C. Expected fragment size of TU encoding ERKKTR after Notl digestion.

    2. TU for MultiTU Plasmids
      The procedure for the generation of TUs for multi-TU plasmids is identical to the one described above for single TUs. However, the connectors used must allow the ordered assembly of the TUs in the final multi-TU plasmid. Below are examples of two separate TUs built from available parts (Figure 4). These two plasmids are assembled in a final plasmid that encodes both transcriptional units (Figure 5B).


      Figure 4. Assembly of two TUs for posterior multi-TU assembly. Plasmids required for first TU (A, ERKKTR) and second TU (B, H2B) assembly. Highlighted in red are LS and RE connectors required for assembly into destination vector. Highlighted in green are connectors that allow the order assembly of first and second TU in the destination vector. Note that L1 connects with R1. Final multi-TU plasmid is shown in Figure 5B.

    3. TU for Multicistronic MTU Plasmids
      Multicistronic plasmids are used when the coexpression of two or more genes of interest is desired. In the MTK, this is achieved by using viral P2A elements. These elements lead the ribosome to skip the synthesis of a peptide bond at the C-terminus of a 2A element, therefore creating two peptides from the same mRNA.
      1. TUs for multicistronic multi-TU plasmids need to be assembled using Part 1 connector plasmids that encode P2A-elements and with Part 2 and Part 4 plasmids that encode spacer sequences that maintain coding-DNA sequences in frame.
      2. The first TU of a multicistronic plasmid must use a Part 1 without P2A elements, a Part 2 that encodes a promoter and a Part 4 without a stop codon and a PA signal (MTK4_006).
      3. The final TU of a multicistronic multi-Plasmid must use a Part 1 encoding a P2A element, a Part 2 that encodes a spacer (MTK2_013) and a Part 4 with a stop codon and a polyadenylation signal.
      4. If more than two TUs are used in the multicistronic construct, the middle TUs, must use Part 1 encoding a P2A element, MTK2_013 and MTK4_006.
      5. The protocol for golden-gate assembly and verification of plasmid is identical to the one detailed above for the single TUs.
    4. TUs for sgRNAs
      TUs encoding sgRNAs are assembled in the same manner as single TUs, but Parts 2, 3 and 4 are replaced by the sgRNA-containing Part 234.

  4. Multi transcriptional unit building
    1. Multi TU units are built from verified TUs that use connectors that will allow them to be assembled, in order, into a destination vector.
    2. Multi TUs are built using the reaction mixture described in Table 7.

      Table 7. Golden Gate reaction setup for MTU assembly


    3. Proceed with Golden Gate reaction as described in Table 4.
    4. Transform bacteria as described in Sharma et al. (2017).
    5. Pick two single white colonies (see Notes) with a sterile microtip and inoculate 5 ml LB media with Kan.
    6. Grow bacteria for 16 h at 37 °C at 250 rpm in a shaker.
    7. Prepare plasmid DNA using Thermo Fisher Miniprep kit, following the manufacturer’s instructions.
    8. Measure the concentration of DNA on a NanoDrop.
    9. Digest plasmids with NotI or other appropriate restriction enzymes, following the manufacturer’s instructions and verify the correct size of fragments.
    10. An example of multi-TU encoding TUs is shown in Figure 4, in the piggybac destination vector (Figure 5).


      Figure 5. Assembly and verification of a Multi-TU encoding ERKKTR and H2B in piggybac destination vector. A. Plasmids required for the assembly of multi-TU. Highlighted in red is destination vector enconding required sequences for piggybac-directed insertion into genome. B. Assembled multi-TU encoding ERKKTR and H2B. Different plasmids used in assembly are shown in different colors and Notl sites. C. Expected fragment size of multi-TU encoding ERKKTR and H2B after Notl digestion.

Notes

Picking white colonies
Correct assemblies into MTK0_027, MTK678_001 or any final destination vector will produce white E. coli colonies after transformation. Most wrong assemblies will generate green colonies. This happens because the destination vectors contain a bacterial GFP-expression cassette that is lost upon correct assembly. While green colonies are visible under white light, they are easily distinguishable under blue light. For that, we use a Gel Documentation System as shown in Figure 6.


Figure 6. Transformants of part domestication. Plate with E. coli transformed with product of Golden Gate Assembly of MTK0_027 and 3 PCR products under (A) white light and (B) blue light. Incorrect assemblies are easily detectable under blue light (green colonies, arrows).

Recipes

  1. T4 DNA ligase + 2.5% PEG
    1. Thaw 10x T4 Ligase buffer
    2. Dilute 50% PEG 1:20 in Ligase buffer
    3. Aliquot 6 μl in PCR microtubes
    4. Store at -20 °C

Acknowledgments

The protocol presented herein was adapted from Fonseca et al. (2019). We would like to thank Seesha Takagashi and Marisa Rosa for critical feedback on the protocol. This work was supported by the National Science Foundation Award # 1715108 to H.E-.S and the Defense Advanced Research Projects Agency (grant number HR0011-16-2-0045 to H.E-.S). H.E-.S is an investigator in the Chan Zuckerberg Biohub. This work was also supported by the National Defense Science & Engineering Graduate (NDSEG) Fellowship awarded to A.R.B..

Competing interests

No competing interests.

References

  1. Akama-Garren, E. H., Joshi, N. S., Tammela, T., Chang, G. P., Wagner, B. L., Lee, D. Y., Rideout, W. M., 3rd, Papagiannakopoulos, T., Xue, W. and Jacks, T. (2016). A modular assembly platform for rapid generation of DNA constructs. Sci Rep 6: 16836.
  2. Duportet, X., Wroblewska, L., Guye, P., Li, Y., Eyquem, J., Rieders, J., Rimchala, T., Batt, G. and Weiss, R. (2014). A platform for rapid prototyping of synthetic gene networks in mammalian cells. Nucleic Acids Res 42(21): 13440-13451.
  3. Fonseca, J. P., Bonny, A. R., Kumar, G. R., Ng, A. H., Town, J., Wu, Q. C., Aslankoohi, E., Chen, S. Y., Dods, G., Harrigan, P., Osimiri, L. C., Kistler, A. L. and El-Samad, H. (2019). A toolkit for rapid modular construction of biological circuits in mammalian cells. ACS Synth Biol.
  4. Halleran, A. D., Swaminathan, A. and Murray, R. M. (2018). Single day construction of multigene circuits with 3G assembly. ACS Synth Biol 7(5): 1477-1480.
  5. He, F. (2011). Standard PCR Protocol. Bio-101: e53.
  6. Lee, M. E., DeLoache, W. C., Cervantes, B. and Dueber, J. E. (2015). A highly characterized yeast toolkit for modular, multipart assembly. ACS Synth Biol 4(9): 975-986.
  7. Martella, A., Matjusaitis, M., Auxillos, J., Pollard, S. M. and Cai, Y. (2017). EMMA: an extensible mammalian modular assembly toolkit for the rapid design and production of diverse expression vectors. ACS Synth Biol 6(7): 1380-1392.
  8. Pérez-González, A., Kniewel, R., Veldhuizen, M., Verma, H. K., Navarro-Rodríguez, M., Rubio, L. M. and Caro, E. (2017). Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria. BMC Biotechnol 17(1): 80.
  9. Pollak, B., Cerda, A., Delmans, M., Álamos, S., Moyano, T., West, A., Gutiérrez, R. A., Patron, N. J., Federici, F. and Haseloff, J. (2019). Loop assembly: a simple and open system for recursive fabrication of DNA circuits. New Phytol 222(1): 628-640.
  10. Sharma, N., Anleu Gil, M. X. and Wengier, D. (2017). A Quick and Easy Method for Making Competent Escherichia coli Cells for Transformation Using Rubidium Chloride. Bio-101: e2590.
  11. Weber, E., Engler, C., Gruetzner, R., Werner, S. and Marillonnet, S. (2011a). A modular cloning system for standardized assembly of multigene constructs. PLoS One 6(2): e16765.
  12. Weber, E., Gruetzner, R., Werner, S., Engler, C. and Marillonnet, S. (2011b). Assembly of designer TAL effectors by Golden Gate cloning. PLoS One 6(5): e19722.

简介

[摘要 ] 快速组装和原型细胞电路的能力对于生物学研究及其在生物技术和医学中的应用至关重要。哺乳动物工具箱(MTK)是基于金门大桥的克隆工具箱,用于快速,可复制和通用的大型DNA载体组装及其在哺乳动物模型中的实现。MTK由精选的,模块化的零件组成的精选库组成,这些零件可以组装成转录单位,并进一步编织成复杂的电路。这些电路很容易重新利用,并通过不同的方法引入哺乳动物细胞。

[背景 ] 分子克隆是现代生物技术与重新利用重组DNA导入多种基因电路可以表示目的的频谱的能力的标志。但是,探索遗传电路构建中可能存在的排列的主要局限性在于能否对电路设计进行快速原型设计,测试和实施改进。为了实现这一目标,需要从常规的克隆方法(如Gibson克隆(Akama-Garren 等人,2016)或限制性酶切消化)中加快从设计遗传回路到将其递送至细胞的时间。我们设计了一个框架,在该框架中,传统的基因电路被分解成其组成部分,以便人们可以轻松地交换这些组成部分,以快速组装出巨大的组合,从而评估每次迭代如何影响功能。受早期克隆工具包迭代的启发(Weber 等人,2011 a和2011b ; Duportet 等人,2014; Lee 等人,2015; Martella 等人,2017;Pérez-González 等人,2017; Halleran 等人,2017)等人,2018年; Pollak 等人,2019年),我们采用了Golden Gate克隆系统,该系统可以通过一锅法将组成部分组装成功能单元(Weber 等人,2011a和2011b )。为了具体实现该框架的前景,我们构建并鉴定了400多种编码多种试剂的DNA片段。我们将策划的库和克隆框架命名为Mammalian ToolKit (MTK)(Fonseca 等,2019)。使用MTK,交换启动子,转换荧光团或快速原型化蛋白质标签构成了在反应中改变一个质粒。这与必须针对每个用例重新设计寡核苷酸和PCR验证正确的装配明显不同,因此不鼓励对目标构建体进行持续优化。该系统的分层性质使部件库可以扩展为转录单位(TU)库,可以进一步组合以创建多转录单位载体,然后将其传递到细胞中。这些TU与交付无关,可促进其多种用途。例如,在通过CRISPR-Cas9同源重组传递到特定基因座的电路中使用的相同TU可以在PiggyBac转座酶转染中再循环以随机整合。从零件验证到交付细胞的初始时间与传统克隆相当(大约4天),而零件和TU的重新用途使电路的大量组合仅用2天即可完成。

尽管MTK具有巨大的优势,但进入壁垒仍然可以阻止那些没有克隆背景的人。为了改善活化能收养所有学科,如何利用现有的库组装新型电路的设计在这里,我们目前的说明,并一步一步的协议“驯养”新的部分(即是,以使它们与金兼容MTK的基于门的组装),组装转录单位(TU),并创建各种多转录单位(multi-TU)。我们的目标是使该工具包可广泛访问和使用,以增强哺乳动物表达系统的开发,以实现生物学上令人兴奋的新发现。

关键字:克隆, 金门克隆, 高通量, Toolkit, 文库, 哺乳动物

材料和试剂


 


无菌尖端为微量(绿- 白TM SPACESAVER TM 堆叠笔芯为的Rainin 移液管)
PCR 微管(BioExpress ,目录号:T-3135-2)
测序引物MTK_P072(gagcctatggaaaaacgc )
测序引物MTK_P073(gggcgtaatttgatatcg )
LB汤(西格玛奥德里奇,目录号:L3022)
LB琼脂肉汤(Lennox)(Sigma-Aldrich,目录号:L2897)
氯霉素(频谱化学,目录号:40310016-2) (工作浓度:100 毫克/ 米升)
羧苄青霉素溶液(频谱化学,目录号:403 10030-2)(工作浓度:25 毫克/ 米升)
硫酸卡那霉素(Sigma-Aldrich公司,目录号:K4000) (工作浓度:100 毫克/ 米升)
T4 DNA 连接酶(NEB,目录号:M0202L)
聚乙二醇(PEG)(Millipore-Sigma,目录号:81268)
T4 多核苷酸激酶(NEB,目录号:M0201L)
T4 DNA 连接酶反应缓冲液(10 x )(NEB,目录号:B0202S)
BsaI-HFv2 (NEB,目录号:R3733S)
FastDigest Esp3I(Thermo Fisher Scientific,目录号:FD0454)
微量准备套件(Thermo Fisher Scientific,目录号:FERK0503PR )
T4 DNA连接酶+ 2.5%PEG(请参阅食谱)
 


设备


 


微量移液器(Rainin Pipet-Lite XLS)
培养箱摇床(新不伦瑞克TM 伊诺? 44,目录编号:M1282-0000)
凝胶文件系统(Protein Simple AlphaImager HP)
热循环仪(Bio -R ad C1000,目录号:1851148)
纳米d ROP (热科学TM 纳米滴TM 2000C ,目录号:ND2000)
程序


 


总览
使用MTK生成遗传回路的过程如图1所示。可以从Addgene (https://www.addgene.org/browse/article/28197510/)订购或从oligos生成MTK库的第1-8部分。和PCR片段。通过BsmBI 金门大会将新零件驯化为对氯霉素具有抗性的目的载体,并选择白色菌落。从零件库中,可以通过将第1-8部分与BsaI Golden Gate反应组装并选择白色,耐羧苄青霉素的克隆来构建转录单位(TU)。TU可以单独或与其他TU一起递送或进一步组合到卡那霉素抗性目的载体中。该组装是通过BsmBI 金门反应完成的,该反应可生成准备引入哺乳动物细胞的质粒。


 


D:\ Reformatting \ 2020-1-6 \ 1902929--1309 Joao Fonseca 828905 \ Figs jpg \ Fig1.jpg


图1. MTK克隆过程概述。A. P 领域中是从寡聚或PCR产物克隆进用氯霉素抗性驯化矢量BsmBI 金门组件。将新克隆的部分或Addgene 提供的库中的部分合并,以产生耐羧苄青霉素的转录单位(TU)。为了递送到哺乳动物细胞中,可以将单个或多个TU进一步组合以产生抗卡那霉素的多个转录单位(Multi-TU)。B.标准TU由8部分组成。第1部分包含5'连接器,第2部分是5'UTR(通常是启动子),第3部分包括编码序列,第4部分是3'UTR(通常是终结器),第5部分包括3'连接器。第6至8部分未显示,通常编码细菌抗性标记。


 


部分d omestication
现有零件
从Addgene 订购零件(https://www.addgene.org/browse/article/28197510/)。
用相应的抗生素在LB琼脂平板中划线细菌。
用无菌微尖端挑出一个白色菌落,并用相应的抗生素接种5 ml LB培养基。
在37处培养细菌16小时 ℃,在250rpm的一个振荡器。
使用的Thermo Fisher Miniprep试剂盒,以下制备质粒DNA 的制造商的说明书。
测量的DNA对的浓度纳米d ROP 。
用引物和MTK_P072 MTK_P073序列的质粒,以确保存在的正确部分。
聚合酶链反应
正向引物设计为(N 4 )CGTCTCNTCGGTCTCN (X 4-6 )(Y 16-22 )。
N是任何基数。


X是特定于零件类型的BsaI 突出碱基。参见表1。


Y是与特定部分同源的碱基,并允许PCR。


反向引物设计为(N 4 )CGTCTCNGGTCTCN (X 4-6 )(Y 16-22 )。
N是任何基数。


X是零件特有的BsaI 突出碱基。参见表1。


Y是与特定部分同源的反向互补碱基,其允许PCR。


从idtDNA (https://www.idtdna.com/)订购引物作为定制的25 nmole DNA寡核苷酸
对于第1部分(连接器),和第5'部分(反向连接器),一个附加的悬垂需要被加入到正向引物(见? 能够2) 。
对于第5部分(连接器),和第1'部分(反向连接器),一个附加的悬垂需要被添加到反向引物(见? 能够2) 。
?F 或部分小号2 ,3 ,4,其为spCAS9编码sgRNAs,以下寡核苷酸被排序为定制25个纳摩尔DNA寡聚物从idtDNA :
TGTTTG(N 20 )G


TAAAC(N” 20 )CA


N是向导特定的序列,而d N'是其反补


 


表1.部分驯化的引物


 


 


 


 


部分


正向引物突出


反向引物突出


1个


CCCTCGTCTCaNNNN


CGTT


1 (反向连接器)


CCCT


CGTTCGTCTCtNNNN


2


咨询委员会


卡塔


3


TATG


加特


3a


TATG


美国汽车协会


3b


TTCT


加特


4


技师


中国计算机学会


4a


空中交通管制


认证证书


4b


泰格


中国计算机学会


5


燃气轮机


TGTACGTCTCtNNNN


5 (反向连接器)


GCTGCGTCTCaNNNN


TGTA


6


塔卡


培训中心


7


加特


泰格


8


中国注册会计师协会


AGGG


8a


中国注册会计师协会


空中交通管制


8b


卡特


AGGG


234


咨询委员会


中国计算机学会


 


表2.特定于连接器的悬垂


连接器


悬垂


LS,LS'


5' CTGA


L1,R1


5' CCAA


L2,R2


5' GATG


L3,R3


5' GTTC


L4,R4


5' GGTA


L5,R5


5' AAGT


L6,R6


5' CCCT


L7,R7


5' GCGG


L8,R8


5' TTTA


RE,RE'


5' AGCA


 


移除内部BsmBI 和BsaI 网站
如果存在内部BsaI 或BsmBI 位点,则需要在这些位点侧翼的其他引物。这些将创建额外的PCR产物,这些产物在金门反应中汇集在一起??,以生成与MTK兼容的部分。需要遵循以下要求:
底漆应正向使用BsmBI 位点,以确保它们不包含在最终产品中。
PCR产生的突出应与零件构建产生的突出(TCGG和GGCT)尽可能不同。例如CATA,而不是TAGG。
去除BsaI / BsmBI 位点的点突变应在编码DNA序列中保持沉默。
去除BsaI / BsmBI 位点的点突变应保持非编码DNA序列中的CG含量。
用于去除ERKKTR 圆顶中内部BsaI 位点的引物示例(ERK激酶易位报道基因,MTK3_021,图2)。悬在了考试PLE是AGGA。
 


D:\ Reformatting \ 2020-1-6 \ 1902929--1309 Joao Fonseca 828905 \ Figs jpg \ Fig2.jpg             


图2. 驯化之前的ERKKTR 序列的一部分。BSAL 网站是突出的黄色。无论正向和反向引物编码BsmBI 网站对后金门总成。此外,反向引物具有一个到? 替代该保持G48,但除去BSAL 从所述最终产品。


 


为了简化编码序列的零件驯化,我们创建了一个python脚本(可在此处找到:https : //github.com/weinerlab/mtk_primer_generator),该脚本可生成给定输入序列和零件编号的候选引物。该脚本会找到需要删除的BsmBI 和BsaI 限制位点,然后生成能够删除每个限制位点的潜在沉默突变列表。最后,它检查这些潜在突变的组合,以确保与Golden Gate组件的一般兼容性以及彼此之间的相互兼容性,从而实现精简的一锅式Golden Gate组件装配。
PCR产生的零件的金门反应
使用表3中所示的反应混合物,将生成的PCR(He,2011 )或基因区块编码部分驯化在PCR微管中。
运行在表4中描述的方案一thermocycl ER。
 


表3.零件驯化的Golden Gate反应设置


 


 


 


 


 


试剂


体积


MTK0_027(50 飞摩尔/ μ 升)


0.5 μ 升


PCR或基因片段块(50 飞摩尔/ μ 升)


0.5 μ 升每个片段的


具有2.5%PEG的T4 DNA连接酶缓冲液(10 x )


1 μ 升


T4 DNA连接酶


0.5 μ 升


Esp3I


0.5 μ 升


蒸馏水


7.5 μ 升- (0.5 μ 升X片段的数量)


 


 


表4.金门反应的热循环条件


温度


持续时间


重复


37°摄氏度


2分钟


24 倍


16°摄氏度


4分钟


37°摄氏度


10分钟


1 个


80°摄氏度


10分钟


1 个


12°摄氏度


保持


1 个


 


如果必须在驯化部分保留内部Bsm bI 位点,则将热循环反应的第三步(表4,37°C,10分钟)从实验方案中删除。
如Sharma 等人所述转化细菌。(2017)。
从步骤B 3进行现有零件的驯化。
一般,挑2个丝毫? 菌落(见注解)中的情况下的PCR增加了一个突变在新克隆的一部分。


寡聚生成零件的金门反应
首先,通过将以下混合物在PCR微管中于37°C孵育1小时,将寡核苷酸生成部分的寡核苷酸磷酸化(表5)。
 


表5. Oligo退火反应设置


试剂


体积


正向寡核苷酸(100 μ 中号)


1 μ 升


反向寡(100 μ 中号)


1 μ 升


T4 DNA连接酶缓冲液(10 x )


1 μ 升


T4 PNK


1 μ 升


蒸馏水


6 μ 升


 


稀释的磷酸化寡核苷酸机智?190 μ 升蒸馏水。
为了使寡核苷酸退火,准备了热循环仪规程以将反应温度在96°C下保持6分钟,然后将每秒0.1°C的温度降至23°C。然后将反应无限期地保持在23℃。
使用表3中所述的反应混合物进行驯化,将PCR片段替换为相同体积的退火寡核苷酸。对于spCAS9 sgRNA驯化,将MTK0_027替换为MTK0_001。
如上所述,从步骤4c到4e 。
 


转录单位(TU)组件
单TU
转录单位(TU)由MTK库的第1至第8部分定义。如果所需的电路仅需要一个TU,我们建议将TU与MTK678_001以及适当的Part 1 LS连接器和Part 5 RE连接器一起安装。这将允许以不同的递送方法将TU回收到哺乳动物细胞中。
使用表6中所述的反应混合物构建单个TU。
如果需要额外的部件(例如,而不是第3部分,使用部分3a和部分3b)中,蒸馏水变化体积,使得反应体积为总等于10 μ 升。


 


表6. TU组装的金门反应设置


试剂


体积


质粒编码部分1与LS连接器(50 飞摩尔/ μ 升)


1 μ 升


质粒编码第2部分(50 飞摩尔/ μ 升)


1 μ 升


质粒编码第3部分(50 飞摩尔/ μ 升)


1 μ 升


质粒编码4部分(50 飞摩尔/ μ 升)


1 μ 升


质粒编码部5与RE连接器(50 飞摩尔/ μ 升)


1 μ 升


质粒编码678部分


(MTK678_001,50 飞摩尔/ μ 升)


0.5 μ 升


具有0.25%PEG的T4 DNA连接酶缓冲液(10 x )


1 μ 升


T4 DNA连接酶


0.5 μ 升


BsaI-HFv2


0.5 μ 升


蒸馏水


2.5 μ 升


 


按表4所述进行金门反应。
如Sharma 等所述转化细菌。(2017年)。
用无菌微量吸头挑出两个单一的白色菌落(见注),并用羧苄青霉素接种5 ml LB培养基。
在生长细菌16小时37℃下在250rpm的一个振荡器。
使用的Thermo Fisher Miniprep试剂盒,以下制备质粒DNA 的制造商的说明书。
测量的DNA对的浓度纳米d ROP 。
消化的质粒用NotI位或其他适当的限制性内切酶小号,按照制造商的指示,并验证片段的正确大小。
TU编码与荧光蛋白mAzamiGreen 融合的ERKKTR 的CAG-d riven 表达的TU 实例,带有bgh 聚腺苷酸化信号(图3)。
 


D:\ Reformatting \ 2020-1-6 \ 1902929--1309 Joao Fonseca 828905 \ Figs jpg \ Fig3.jpg


图3.单个TU编码ERKKTR的组装和验证。A.组装TU以表达mAzamiGreen 融合的ERKKTR 所需的质粒。质粒enconding 连接器LS和RE(以红色突出显示)是用于单个TU电路的生成是必不可少的。B.组装的ERKKTR编码TU。组装中使用的不同零件以不同的颜色和Notl 位置显示。C.预期后编码ERKKTR TU的片段大小的NotI 消化。


 


TU用于MultiTU 质粒
用于多TU质粒的TU的生成过程与上述用于单个TU的方法相同。? H但是,所用必须允许连接器中相应的最终的多TU质粒有序组件中的TU的。下面是从可用零件构建的两个独立TU的示例(图4)。这两个质粒组装在编码两个转录单位的最终质粒中(图5B)。


 


D:\ Reformatting \ 2020-1-6 \ 1902929--1309 Joao Fonseca 828905 \ Figs jpg \ Fig4.jpg


图4.两个TU的组装,用于后部多TU组装。第一个TU(A,ERKKTR)和第二个TU(B,H2B)组装所需的质粒。红色突出显示的是组装到目标向量中所需的LS和RE 连接器。绿色突出显示的连接器允许在目标向量中对第一和??第二TU进行有序组装。请注意,L 1 与R1连接。最终的多TU质粒如图5B所示。


 


 


TU用于多顺反子MTU质粒
当需要两个或多个目的基因的共表达时,可使用多顺反子质粒。在MTK中,这是通过使用病毒P2A元素实现的。这些元素导致核糖体跳过2A元素C端的肽键合成,因此从相同的mRNA产生两个肽。


TU的用于多顺反多TU质粒需要使用部分被组装1连接器的质粒编码P2A元素和与部分2和部分4的质粒小号其编码间隔区序列维持在帧编码-DNA序列。
多顺反子质粒的第一个TU 必须使用不含P2A 元件的第1 部分,编码启动子的第2部分和不含终止密码子和PA信号(MTK4_006)的第4 部分。
多顺反子多质粒的最终TU 必须使用编码P2A元素的第1部分,编码间隔子(MTK2_013)的第2部分和带有终止密码子和聚腺苷酸化信号的第4 部分。
如果在多顺反子结构中使用了两个以上的TU ,则中间TU必须使用第1 部分编码的P2A元素MTK2_013和MTK4_006。
金门组装和质粒验证的协议与上面针对单个TU详述的协议相同。
sgRNA的TU
编码sgRNA的TU的组装方式与单个TU相同,但第2、3和4部分被含sgRNA的234 部分代替。


 


多牛逼ranscriptional ü 尼特b uilding
多个TU单元由经过验证的TU构建而成,这些TU使用连接器将其按顺序组装到目标向量中。
使用表7中描述的反应混合物构建多个TU 。
 


表7. MTU组装的金门反应设置


试剂


体积


质粒编码的TU (50 飞摩尔/ μ 升)


1 μ 升每


质粒编码PART0 (目的载体,50 飞摩尔/ μ 升)


0.5 μ 升


具有2.5%PEG的T4 DNA连接酶缓冲液(10 x )


1 μ 升


T4 DNA连接酶


0.5 μ 升


Esp3I


0.5 μ 升


蒸馏水


7.5 - (0.5 μ 升X TU的质粒的数量)μ 升


 


按表4所述进行金门反应。
如Sharma 等所述,转化细菌。(2017年)。
用无菌微尖端挑出两个单一的白色菌落(请参阅“注释”),并用Kan接种5 ml LB培养基。
在37℃下生长的细菌16小时在250rpm的一个振荡器。
使用的Thermo Fisher Miniprep试剂盒,以下制备质粒DNA 的制造商的说明书。
测量该DNA的上的浓度纳米d ROP。
消化的质粒用NotI位或其他适当的限制性内切酶小号,按照制造商的指示,并验证该片段的正确大小。
电子多TU编码TU的xample 被示出在图4中,在piggyBac转目的地向量(图5)。
 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ 2020-3-05 \ 1902929--1309 Joao Fonseca 828905 \ Figs jpg \ Fig5-udated.jpg


图5.在piggybac 目标向量中编码ERKKTR和H2B的Multi-TU的组装和验证。所需的组件多-T的A.质粒在红色U.突出显示的是目的载体enconding 为所需的序列piggyBac转-directed插入基因组中。B.组装的多TU编码ERKKTR和H2B。组装中使用的不同质粒以不同的颜色和NotI位点显示。多TU的C.预期片断大小后编码ERKKTR和H2B NotI位消化。


 


笔记


 


采摘白色殖民地


正确组件插入MTK0_027,MTK678_001或任何最终目的地向量会产生白色E. 大肠杆菌转化后的菌落。大多数错误的程序集都会生成绿色菌落。发生这种情况是因为目的载体包含正确表达后会丢失的细菌GFP表达盒。尽管在白光下可见绿色菌落,但在蓝光下很容易区分它们。为此,我们使用了图6所示的凝胶文档系统。


 


D:\ Reformatting \ 2020-1-6 \ 1902929--1309 Joao Fonseca 828905 \ Figs jpg \ Fig6.jpg


图6.部分驯化的转化体。板与大肠杆菌与MTK0_027的金门装配和3的PCR产物的UND产物转化ER (A)白光和(B)的蓝色光。在蓝光下(绿色菌落,箭头)很容易检测到不正确的组件。


 


菜谱


 


T4 DNA连接酶+ 2.5%PEG
解冻10x T4连接酶缓冲液
在连接酶缓冲液中稀释50%PEG 1:20
等分试样6 μ升在PCR微管
储存在-20 °C
 


致谢


 


本文提供的协议改编自Fonseca 等。(2019)。我们要感谢Seesha Takagashi 和Marisa Rosa对协议的重要反馈。这项工作得到了HE-.S 的国家科学基金会奖#1715108和国防高级研究计划局的资助(HE-.S的资助号HR0011-16-2-0045 )。HE-.S是Chan Zuckerberg Biohub 的调查员。这项工作也得到了授予ARB的国防科学与工程研究生(NDSEG)奖学金的支持。


 


 


利益争夺


 


没有利益冲突。


 


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引用:Fonseca, J. P., Bonny, A. R., Town, J. and El-Samad, H. (2020). Assembly of Genetic Circuits with the Mammalian ToolKit. Bio-protocol 10(5): e3547. DOI: 10.21769/BioProtoc.3547.
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