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Design and Direct Assembly of Synthesized Uracil-containing Non-clonal DNA Fragments into Vectors by USERTM Cloning
设计并通过USERTM克隆直接将合成的含尿嘧啶非克隆DNA片段装配到载体中   

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Abstract

This protocol describes how to order and directly assemble uracil-containing non-clonal DNA fragments by uracil excision based cloning (USER cloning). The protocol was generated with the goal of making synthesized non-clonal DNA fragments directly compatible with USERTM cloning. The protocol is highly efficient and would be compatible with uracil-containing non-clonal DNA fragments obtained from any synthesizing company. The protocol drastically reduces time and handling between receiving the synthesized DNA fragments and transforming with vector and DNA fragment(s).

Keywords: USERTM cloning(USERTM克隆), Cloning of synthesized non-clonal DNA fragments(克隆合成的非克隆DNA片段), Fusion of DNA fragments(DNA片段融合), Uracil excision based cloning(基于尿嘧啶切除的克隆), uNCDFs(uSTRINGs), Geneart(Geneart)

Background

For synthesized DNA, non-clonal linear DNA fragments (NCDF) have emerged as a cheaper and faster alternative to clonal fragments that are delivered sequenced and in a circular vector. NCDFs can be regarded as an IKEA solution to DNA synthesis where it is the customers that assemble their DNA fragments into a vector of choice and subsequently must verify the sequence of the final construct. In this protocol, we obtained uracil-containing NCDFs from Thermo Fisher Scientific Geneart, whose NCDFs are termed DNA strings. The uracil-containing NCDFs will here be named uNCDFs. In uNCDFs, uracils are inserted at designated positions during synthesis. We were able to clone uNCDFs directly into USER cloning compatible vectors by simply re-suspending the fragments in water, incubating them with linearized USER vector and USER enzyme (as described below) followed by transformation of E. coli. The procedure requires minimal handling, lasts less than 1 h from receipt to transformation and requires very small amounts of DNA. We have tested uNCDFs for almost a year in our laboratory and found them to perform consistently well. We recently cloned 13 transporter genes into a USER compatible Xenopus oocyte expression vector. Each gene fragment was obtained as a uNCDF (~2 kb in size). We tested a single colony for each cloning and found that 12 of the genes were inserted correctly into the pNB1u destination vector. For the last clone we found a single mutation which was correct in the second colony that we tested for that fragment (Jorgensen et al., 2017). The pNB1u vector is approximately 2.5 kb in size. In another project, we cloned a larger batch of uNCDFs composed of 64 fragments 1-2.4 kb in size into a variety of USER compatible vectors including the large (~10 kb) USER compatible pCambia based vectors (Nour-Eldin et al., 2006). Of these, 55 were correct after testing a single colony, 62 were correct after sequencing a second colony, while the remaining two were found in a correct form in the third colony (unpublished). We also used the protocol to seamlessly fuse three fragments together and insert them into a destination vector by USER fusion (Geu-Flores et al., 2007). Thus, we experienced high efficiency when cloning uNCDFs which was comparable to the efficiency of cloning uracil-containing DNA fragments generated via PCR. Moreover the uNCDFs were shown to work well for both small and large USER compatible vectors.

The innovation in this protocol is the ability to design and order synthesized DNA fragment containing uracils at appropriate locations. To appreciate the value of this advance and ensure an understanding of this cloning technique for researchers who have not used USER cloning previously, a brief introduction to USER cloning is given below.

The principle behind the efficiency of USER cloning lies in the ability to generate long, complementary overhangs. These overhangs can anneal to form stable hybridization products that can be used to transform E. coli without prior ligation. Most importantly, their generation is not dependent on the introduction of restriction sites. Hitherto, the generation of long single stranded overhangs has proceeded in two steps. First, a PCR reaction was performed on a DNA fragment of interest using primers containing a short 8-16 nt upstream extension that is preceded by a single deoxyuridine residue. The resulting PCR products are treated briefly with a commercial mix of uracil DNA glycosylase and DNA glycosylaselyase Endo VIII. These enzymes, are included in the USERTM enzyme mix, and remove the two single deoxyuridine residues and enable the dissociation of the short, single-stranded fragments lying upstream from the cleavage sites. For the generation of overhangs in a USER compatible destination vector, a short cassette is inserted into it so that digestion with a restriction- and a nicking enzyme creates the desired long overhangs. The fact that long, custom-made overhangs can be generated on PCR products can be exploited to generate a series of PCR products with complementary overhangs. This enables the generation of a hybridization product consisting of a vector and multiple PCR products, which can be fused into a compatible vector as easily as a single PCR product. For more detailed information on the cloning method, readers are referred to the following references on how to perform USER cloning and USER fusion (Nour-Eldin et al., 2006 and 2010; Geu-Flores et al., 2007). Additionally, bioinformatic tools have been developed to aid design of primers and strategy of USER cloning and fusion including the (Automated DNA Modifications with USER cloning) AMUSER web server tool for automated primer design (Genee et al., 2015) and an interactive lab simulation that teaches the principles of USER cloning (https://www.labster.com/simulations/user/). Finally, overlap design for USER fusion has recently been optimized (Cavaleiro et al., 2015).

Introducing uracils into DNA fragments was hitherto performed via PCR using uracil-containing primers. The PCR was performed after receiving the NCDF and required designing and ordering of appropriate uracil-containing primers. Incorporating uracils in DNA fragments during synthesis omits the need to design and order uracil-containing primers and also omits the need to perform a PCR reaction, this drastically reduces time and handling between receiving the synthesized DNA fragments and transforming with vector and DNA fragment(s).

Materials and Reagents

  1. For ordering uracil-containing NCDFs
    1. Sequence of DNA to be synthesized (optimized for desired organism)
      Note: Depending on the fidelity of synthesis, any size of fragment is in principle suitable for synthesis. Until now the maximum size of NCDFs we have been able to order is 3 kb (varies depending on the company) and the fidelity in terms of how many colonies we had to sequence to find a correct clone has generally been 1-2.
    2. Sequence of USER tails to be added for insertion into USER vector (for single gene insertion)
    3. Sequence of USER overlaps for seamless USER fusion
    Note: For insertion into USER compatible vectors, the USER tails are given by the USER cassette that has been inserted. Please consult the relevant publications. For USER fusion we typically use USER overlaps between 7-16 bp in length.

  2. For linerarizing USER compatible vector
    1. 1.5 ml centrifuge tubes (Eppendorf tubes)
    2. USER compatible vector (Nour-Eldin et al., 2006 and 2010; Geu-Flores et al., 2007)
    3. PacI restriction enzyme (New England Biolabs, catalog number: R0547S )
    4. Nt.BbvCI nicking enzyme (New England Biolabs, catalog number: R0632S )
    5. Cutsmart buffer (included when ordering enzymes above)
    6. H2O
    7. PCR purification kit of choice (e.g., QIAquick PCR purification kit) (QIAGEN, catalog number: 28104 )
    Note: For part B, follow protocol published previously (Nour-Eldin et al., 2010).

  3. For cloning uNCDFs into linearized USER compatible vector
    1. 1.5 ml centrifuge tubes (Eppendorf tubes)
    2. CaCl2 competent E. coli cells (DH5α or NEB10β or other common cloning strains)
      Note: Do not perform transformation by electroshocking as the shock will cause the vector and uNCDF to dissociate.
    3. uNCDFs from synthesis company (in this case from Thermo Fisher Scientific Geneart)
      Note: Despite their success, uNCDFs are not yet offered officially by Geneart. Please contact e.g., Anja Martinez at Thermofisher Geneart (anja.martinez@thermofisher.com) or us for ordering questions.
    4. H2O
    5. Linearized USER compatible vector
      Note: A long list of vectors has been made USER cloning compatible for almost all organisms. Search within these three papers (Nour-Eldin et al., 2006 and 2010; Geu-Flores et al., 2007) and the many papers citing them. Please note, we are currently in the process of gathering USER vectors from groups from all over the world in order to deposit them at Addgene for easy accessibility.
    6. USER enzyme (New England Biolabs, catalog number: M5505S )
    7. 5x or 10x PCR buffer (any kind)

Equipment

  1. 37 °C heat block
  2. 42 °C heat block/water bath

Procedure

  1. Design and order uracil-containing NCDFs for single fragment USER cloning
    For cloning single uNCDFs into USER compatible vectors that have been generated by our lab, our generic 5’ and 3’ user tails are added to termini of the fragment to be inserted (5’ USER tail: 5’-GGCTTAAU, 3’ USER tail: 5’-GGTTTAAU) (for inserting into other USER compatible vectors please use the corresponding USER tails). When ordering synthesized DNA we typically codon optimize the sequence for the organism wherein the gene will be expressed. In an example below, we show how we ordered the gene AT1G15210 as a uNCDF from Thermo Fisher Scientific Geneart. As instructed by Geneart, we provided them with a word file containing sequences to be synthesized in FASTA format. At the beginning of the word document, we included general instructions to the synthesis company, which are given in italics below
    Dear synthesis company:
    Green marks a T in the top strand that must be replaced by a U. Turquoise marks an A, which is complementary to a T on the opposite strand that must be replaced by a U (i.e., it is the T on the opposite strand that must be replaced by a uracil). Yellow marks sequence that may not be altered. Non-marked sequence represents coding sequence, which may be changed to overcome synthesis challenges. The gene will be expressed in Xenopus laevis oocytes. Please use the appropriate codon preference table for codon optimization and alternations.



    Note: Punctuation marks approx. 2 kb of sequence that was included in the order but which has been omitted here for brevity. In this example, yellow sequence represents our standard 5’ and 3’ USER tails.

  2. Cloning single uNCDFs into linearized USER compatible vector


    Figure 1. Overview of the USER cloning technique. A USER cassette in a USER-compatible vector (upper left corner) with a restriction site (PacI, light blue) in the middle, one variable nt surrounding it (different for each side, yellow and green), and oppositely oriented nicking sites (Nt.BbvCI, tan). The USER vector is digested with PacI and Nt.BbvCI to generate 8 nt overhangs. A DNA fragment (upper right corner) with uracils at appropriate positions can be generated by PCR as described previously or via synthesis as a uNCDF as described here. Fragment and vector are mixed with USERTM enzyme mix (excising deoxyuridines, pink) and the digested USER-compatible vector. Following brief incubation, the hybridized product can be used to transform E. coli without prior ligation. This figure has been reproduced with permission from (Nour-Eldin et al., 2006).

    1. Each fragment is to be surrounded by USER tails that enable insertion into a USER compatible vector. In this example, we use our X. laevis expression vector pNB1u (Nour-Eldin et al., 2006). uNCDFs are mixed directly with the digested pNB1u vector without prior PCR amplification (Figure1).
      1. Dilute each uNCDF to 100 ng/μl in H2O (or TE buffer pH 8).
      2. The USER-compatible pNB1u X. laevis oocyte expression vector is digested with PacI/Nt.BbvCI overnight, PCR purified and diluted to a concentration of ~50 ng/μl (as previously described [Nour-Eldin et al., 2006 and 2010]).
        Note: If a USER compatible vector is not available, it is possible to generate them via PCR. In that case, the vector backbone is treated as a DNA fragment to be assembled with DNA fragment of interest via USER fusion (please see below).
      3. For the USER reaction, mix 100 ng uNCDF with 50 ng digested pNB1u, 1 U USER enzyme (NEB), 2 µl 5x PCR reaction buffer and 5 µl H2O.
      4. Incubate the reaction at 37 °C for 25 min, and then for 25 min at room temperature.
      5. Transform 50 µl chemically competent E. coli cells with the reaction mixture by heat shock (5 min on ice, 30-45 sec at 42 °C and 5 min on ice).
      6. Plate the transformation mixture on LB-plates containing appropriate antibiotic selection (for pNB1u we use carbenicillin or ampicilin–containing LB plates).
      7. Select three colonies from the plates and grow overnight. Extract plasmids and analyze the extracted plasmids by gel-electrophoresis. Sequence the plasmids with an insert.

  3. Design and order uracil-containing NCDFs for USER fusion of multiple fragments
    For fusion of multiple uNCDFs into USER compatible vectors overlap regions have to be selected and included in adjacent fragments. Overlap regions are selected as previously described by finding a T on the bottom strand and a T on the top strand 7-15 bases downstream of the first T. The pair of selected Ts should be within 20-30 bases distance to the junction site (Figure 2) (Geu-Flores et al., 2007; Nour-Eldin et al., 2010). These Ts will be the ones replaced by a U during synthesis. For insertion into the USER compatible vector, our generic 5’ and 3’ user tails are added to the terminal fragments. When ordering synthesized DNA we typically codon optimize the sequence for the organism wherein the gene will be expressed. In an example below, we show how we designed and ordered three fragments to be fused and inserted into a USER compatible vector by USER cloning. The full-length sequence (~7,200 bp) was split into three fragments (FAS1_A, FAS1_B and FAS1_C). The sequence of their termini is given below. In each fragment, the punctuation in the middle denotes approximately 2 kb of sequence, which was included in the order but has been omitted here for brevity. Each fragment was designed to include overlap regions to the adjacent fragment close to the junction sites. Please see below and see (Geu-Flores et al., 2007; Nour-Eldin et al., 2010) for more detailed information on how to design USER fusion overlap regions. Alternatively, the Amuser web server tool can deisgn overlap regions for any USER fusion approach (Genee et al., 2015). As instructed by Geneart we provided them with a Word file containing sequences to be synthesized in FASTA format. At the beginning of the Word document, we included general instructions to the synthesis company, which are given in italics below.
    Dear synthesis company:
    Green marks a T in the top strand that must be replaced by a U. Turquoise marks an A, which is complementary to a T on the opposite strand that must be replaced by a U (i.e., it is the T on the opposite strand that must be replaced by a uracil). No alterations are allowed in any color shaded sequence. Non-marked sequence represents coding sequence, which may be changed to overcome synthesis challenges. The genes will be expressed in Xenopus laevis oocytes. Please use the appropriate codon preference table for codon optimization and alternations.



    Note: Only the sense strand is included in the ordering process but the result is a uracil-containing double stranded fragment. No alterations are allowed in any colored region during the codon optimization process. Yellow marks our standard 5’ USER tail. Green marks the T, which must be synthesized as U. Turquoise marks the A, whose complimentary T must be synthesized as U. Grey marks the overlap which will be exposed as a single stranded fragment upon USER treatment and which will hybridize to the complimentary single stranded overhang on the adjacent fragment (in this case FAS1_B). Once synthesized, the double stranded FAS1_A uNCDF will look as follows:



    Upon treatment with USER enzyme the double stranded FAS1_A uNCDF will loose sequences lying upstream of the uracils and look as follows:



    As for FAS1_A only the sense strand is included in the ordering process. Green marks the T, which must be synthesized as U. Turquoise marks the A, whose complimentary T must be synthesized as U. Grey marks the overlaps which will be exposed as single stranded overhangs upon USER treatment and which will hybridize to the complimentary single stranded overhangs on the adjacent fragments (in this case the left ‘grey’ will hybridize to the overhang generated on FAS1_A, whereas the right ‘grey’ will hybridize to the overhang generated on FAS1_C). Once synthesized, the double stranded FAS1_B uNCDF will look as follows:



    Upon treatment with USER enzyme the double stranded FAS1_B uNCDF will loose sequences lying upstream of the uracils and look as follows:



    As for FAS1_A only the sense strand is included in the ordering process. Green marks the T, which must be synthesized as U. Turquoise marks the A, whose complimentary T must be synthesized as U. Grey marks the overlap, which will be exposed as single stranded overhang upon USER treatment and which will hybridize to the complimentary single stranded overhangs on the adjacent fragment (in this case the rightmost ‘grey’ overlap region on FAS1_B). Once synthesized, the double stranded FAS1_B uNCDF will look as follows:



    Upon treatment with USER enzyme the double stranded FAS1_C uNCDF will loose sequences lying upstream of the uracils and look as follows:



  4. Fusion and clone of multiple uNCDFs into linearized USER compatible vector


    Figure 2. Overview of the USER fusion technique. Fragments X1, X2, and X3 are to be fused together. Overlap regions are marked in grey. Uracils can be inserted via PCR or as described here during synthesis. The DNA fragments are mixed with a pre-digested USER-compatible vector and treated with the deoxyuridine-excising USERTM enzyme mix. This generated 3’ overhangs that complement each other (indicated by arrows), while the outermost ones complemented the overhangs of the pre-digested vector. This design enables the formation of a stable circular hybridization product that can be transformed directly into E. coli without prior ligation. This figure has been reproduced with permission from (Geu-Flores et al., 2007).

    For fusing and cloning multiple uNCDFs into USER compatible vectors that have been generated by our lab, our generic 8 bp long 5’ and 3’ user tails are added to termini of the terminal fragments. At junction sites uracil-containing overlaps were included to allow seamless fusion upon mixing. When possible we strive to choose fusion overlaps of different lengths and composition to minimize mis-hybridization between fragments. In this example, we fuse and clone three fragments into our X. laevis expression vector pNB1u (Nour-Eldin et al., 2006). Each fragment contained a uracil at the appropriate location in each USER tail. The uracil was incorporated during synthesis. Thus, uNCDFs are mixed in equimolar ratios directly with the digested pNB1u vector without prior PCR amplification with uracil-containing primers.
    1. Dilute each uNCDFs to 50 ng/µl in H2O.
    2. The USER-compatible pNB1u X. laevis oocyte expression vector is digested with PacI/Nt.BbvCI overnight, PCR purified and diluted to a concentration of ~50 ng/µl (as previously described (Nour-Eldin et al., 2006 and 2010)).
    3. For the USER reaction, mix 100 ng of each uNCDFs with 50 ng digested pNB1u, 1 U USER enzyme (NEB), 2 µl 5x PCR reaction buffer and H2O to bring the total volume to 10 µl.
    4. Incubate the reaction at 37 °C for 25 min, and then for 25 min at room temperature.
    5. Transform 50 µl chemically competent E. coli cells (for example NEB10β by heat shock) with the reaction mixture (5 min on ice, 30-45 sec at 42 °C and 5 min on ice).
      Note: Typically the volume of added USER reaction mixture should not exceed 10% of the competent E. coli volume (i.e., max 5 µl reaction mixture to 50 µl E. coli volume, 10 µl reaction mixture to 100 µl E. coli volume etc. Exceeding this ration can reduce transformation efficiency.
    6. Plate the transformation mixture on LB-plates containing appropriate antibiotic selection (for pNB1u we used carbenicillin–containing LB plates).
    7. Select eight colonies from the plates and grow overnight. Extract plasmids and analyze the extracted plasmids by gel-electrophoresis. Sequence the plasmids with an insert.

Notes

If insertion does not succeed in the first trial, we encourage users–in addition to the USER cloning–to clone the uNCDFs into a blunt-ended cloning vector (such as pJET) for safekeeping and future work.

Acknowledgments

MEJ is supported by a grant from the Danish Council for Independent Research: DFF–6108-00122. DX, AP, CW, DV, SKL and HHN were funded by DNRF99 grant from the Danish National Research Foundation. ZNB was funded by Innovationfund Denmark J.nr.: 76-2014-3 and NW was funded by Human Frontier Science Program RGY0075/2015. The authors declare no conflicts of or competing interest.

References

  1. Cavaleiro, A. M., Kim, S. H., Seppala, S., Nielsen, M. T. and Norholm, M. H. (2015). Accurate DNA assembly and genome engineering with optimized uracil excision cloning. ACS Synth Biol 4(9): 1042-1046.
  2. Genee, H. J., Bonde, M. T., Bagger, F. O., Jespersen, J. B., Sommer, M. O., Wernersson, R. and Olsen, L. R. (2015). Software-supported USER cloning strategies for site-directed mutagenesis and DNA assembly. ACS Synth Biol 4(3): 342-349.
  3. Geu-Flores, F., Nour-Eldin, H. H., Nielsen, M. T. and Halkier, B. A. (2007). USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Res 35(7): e55.
  4. Jorgensen, M. E., Xu, D., Crocoll, C., Ramirez, D., Motawia, M. S., Olsen, C. E., Nour-Eldin, H. H. and Halkier, B. A. (2017). Origin and evolution of transporter substrate specificity within the NPF family. Elife 6.
  5. Nour-Eldin, H. H., Geu-Flores, F. and Halkier, B. A. (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories. Methods Mol Biol 643: 185-200.
  6. Nour-Eldin, H. H., Hansen, B. G., Norholm, M. H., Jensen, J. K. and Halkier, B. A. (2006). Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Res 34(18): e122.

简介

该协议描述如何通过基于尿嘧啶切除的克隆(USER克隆)命令并直接组装尿嘧啶非克隆DNA片段。 该方案的目的是制备与USER TM克隆直接兼容的合成非克隆DNA片段。 该协议是高效率,并将与从任何合成公司获得尿嘧啶非克隆DNA片段相容。 该方案大大减少了接收合成的DNA片段和用载体和DNA片段转化之间的时间和处理。

【背景】对于合成的DNA,非克隆线性DNA片段(NCDF)已经成为更廉价和更快速的替代克隆片段的方法,并且在循环载体中被测序。 NCDF可以被认为是IKEA DNA合成的解决方案,在这个解决方案中,客户将他们的DNA片段组装成一个选择的载体,随后必须验证最终构建体的序列。在这个协议中,我们从Thermo Fisher Scientific Geneart获得含有尿嘧啶的NCDF,其NCDF被称为DNA链。这种含尿嘧啶的NCDF在这里将被命名为uNCDF。在uNCDF中,尿嘧啶在合成过程中插入到指定位置。我们能够通过简单地将片段重悬于水中,将它们与线性化的USER载体和USER酶(如下所述)一起温育,接着转化E,从而将uNCDF直接克隆到USER克隆兼容载体中。大肠杆菌。该程序需要最少的处理,从接收到转化持续不到1小时,并且需要非常少量的DNA。我们在实验室测试了uNCDF近一年,发现它们表现一贯良好。我们最近将13种转运蛋白基因克隆到一个用户兼容的非洲爪蟾卵母细胞表达载体中。每个基因片段以uNCDF(〜2kb大小)获得。我们测试了每个克隆的单个菌落,发现12个基因正确插入到pNB1u目的载体中。对于最后的克隆,我们在第二个菌落中发现了一个正确的突变,我们测试了这个片段(Jorgensen等人,2017)。 pNB1u载体的大小约为2.5kb。在另一个项目中,我们克隆了大量由大小为1-2.4kb的64个片段组成的更大批量的uNCDF,并将其包含到大量(〜10kb)用户兼容的基于pCambia的载体(Nour-Eldin等人。,2006)。其中55个是单个菌落检测后正确的,62个是第二个菌落测序后正确的,而另外两个是在第三个菌落(未发表)中以正确形式发现的。我们还使用该协议将三个片段无缝融合在一起,并通过USER融合将它们插入目的载体(Geu-Flores等人,2007)。因此,我们在克隆uNCDF时经历了高效率,这与通过PCR产生的含尿嘧啶的DNA片段的克隆效率相当。此外,uNCDF被证明适用于小型和大型USER兼容的载体。

本协议的创新之处在于能够在合适的位置设计和订购含有尿嘧啶的合成DNA片段。为了体会这种进步的价值,并确保对以前没有使用过USER克隆的研究人员了解这种克隆技术,下面给出对USER克隆的简要介绍。

USER克隆效率的原理在于能够产生长长的互补突出。这些突出端可以退火形成可用于转化E的稳定的杂交产物。无需预先结扎。最重要的是,他们这一代并不依赖于限制性地点的引入。迄今为止,长单一悬挑的产生分两步进行。首先,使用含有短8-16nt上游延伸的引物(其前面是单个脱氧尿苷残基)对目的DNA片段进行PCR反应。所得PCR产物用尿嘧啶DNA糖基化酶和DNA糖基化酶Endo VIII的商业混合物简单处理。这些酶被包含在USER TM TM酶混合物中,并除去两个单脱氧尿苷残基,并使位于切割位点上游的短的单链片段解离。为了在用户兼容的目标载体中产生突出端,将短的盒插入其中,以便用限制酶和切口酶消化产生所需的长突出端。可以在PCR产物上产生长而定制的突出端的事实可用于产生具有互补突出端的一系列PCR产物。这使得能够产生由载体和多个PCR产物组成的杂交产物,其可以像单个PCR产物那样容易地融合到相容的载体中。更多关于克隆方法的详细信息,关于如何执行USER克隆和用户融合(Nour-Eldin等人,2006和2010; Geu-Flores等人。,2007)。此外,已经开发了生物信息学工具来帮助设计USER克隆和融合的引物和策略,包括(用户克隆的自动化DNA修饰)用于自动引物设计的AMUSER网络服务器工具(Genee等人, 2015)和一个交互式实验室模拟,教导用户克隆的原则( https://www.labster.com /模拟/用户/ )。最后,用于USER融合的重叠设计最近已经被优化(Cavaleiro et al。,2015)。

迄今通过使用含尿嘧啶的引物进行PCR将尿嘧啶导入DNA片段。在接受NCDF后进行PCR,并且需要设计和排序合适的含尿嘧啶的引物。在合成过程中将尿嘧啶结合到DNA片段中省略了设计和排序含尿嘧啶的引物的需要,也省去了进行PCR反应的需要,这大大缩短了接收合成的DNA片段和用载体和DNA片段转化的时间和处理)。

关键字:USERTM克隆, 克隆合成的非克隆DNA片段, DNA片段融合, 基于尿嘧啶切除的克隆, uSTRINGs, Geneart

材料和试剂

  1. 用于订购含尿嘧啶的NCDF
    1. 要合成的DNA序列(针对所需有机体进行优化)
      注:根据合成的保真度,任何大小的片段原则上都适合合成。到目前为止,我们已经能够订购的NCDF的最大尺寸是3kb(根据公司的不同而不同),并且在我们不得不为了找到正确的克隆而需要测序的殖民地的保真度一般是1-2。
    2. 用户尾巴序列添加插入USER向量(单基因插入)
    3. 用户重叠的序列无缝USER融合
    注:为了插入USER兼容矢量,USER尾部由已经插入的USER盒提供。请查阅相关出版物。对于USER融合,我们通常使用7-16 bp长的USER重叠。

  2. 用于分类USER兼容的矢量
    1. 1.5毫升离心管(Eppendorf管)
    2. USER兼容的载体(Nour-Eldin等人,2006和2010; Geu-Flores等人,2007)。
    3. PacI限制酶(New England Biolabs,目录号:R0547S)
    4. Nt。Bbv CI切口酶(New England Biolabs,目录号:R0632S)
    5. Cutsmart缓冲液(包括在订购酶时)
    6. H <2> O
    7. PCR纯化试剂盒(例如,QIAquick PCR纯化试剂盒)(QIAGEN,目录号:28104)
    注:对于B部分,请遵循之前发布的协议(Nour-Eldin等,2010)。

  3. 将uNCDF克隆到线性化的USER兼容向量中
    1. 1.5毫升离心管(Eppendorf管)
    2. CaCl 2 2感受态大肠杆菌细胞(DH5α或NEB10β或其他常见的克隆菌株)
      注意:电击不要进行转换,因为电击会导致载体和uNCDF分离。
    3. 来自合成公司的uNCDF(在这种情况下来自Thermo Fisher Scientific Geneart)
      注:尽管他们成功,uNCDFs还没有由Geneart正式提供。请联系,例如Anja Martinez在GeneFactory( anja.martinez@thermofisher。 )或我们订购问题。
    4. H <2> O
    5. 线性化的USER兼容矢量
      注意:一个很长的载体列表已经使USER克隆几乎适用于所有生物体。在这三篇论文(Nour-Eldin等人,2006和2010; Geu-Flores等人,2007)以及许多论文中引用他们。请注意,我们目前正在收集来自世界各地的用户群,以便将它们存放在Addgene以方便访问。
    6. USER酶(新英格兰生物实验室,目录号:M5505S)
    7. 5x或10x PCR缓冲液(任何种类)

设备

  1. 37°C的热块
  2. 42°C加热块/水浴

程序

  1. 设计和命令含尿嘧啶的NCDF用于单片段USER克隆
    为了将单个uNCDF克隆到由我们的实验室生成的USER兼容的载体中,我们的通用5'和3'用户尾部被添加到要插入的片段的末端(5'USER尾:5'-GGCTTAAU,3'USER尾:5'-GGTTTAAU)(用于插入其他USER兼容的载体,请使用相应的USER尾部)。当命令合成的DNA时,我们通常密码子优化其中基因将被表达的生物体的序列。在下面的例子中,我们展示了我们如何订购基因AT1G15210作为Thermo Fisher Scientific Geneart的uNCDF。按照Geneart的指示,我们向他们提供了一个包含要用FASTA格式合成的序列的文件。在word文档的开始处,我们包含了对合成公司的一般说明,这些说明在下面用斜体表示 亲爱的合成公司:
    绿色标志T必须替换为U.绿松石标志着一个A,它与另一个T上的T互补,必须被U取代(即它是T上的T必须由尿嘧啶取代的相反的链)。黄色的标记序列可能不会被改变。未标记的序列代表编码序列,其可以被改变以克服合成挑战。该基因将在非洲爪蟾卵母细胞中表达。请使用适当的密码子偏好表进行密码子优化和改变。



    注:标点符号约。序列中包含的2kb序列,但为简洁起见,此处省略。在这个例子中,黄色序列表示我们的标准5'和3'用户尾巴。

  2. 将单个uNCDF克隆到线性化的USER兼容向量中


    图1. USER克隆技术概述。 在用户兼容的矢量(左上角)中的用户暗盒,在中间有一个限制位点( Pac I,浅蓝色),一个包围它的变量nt(每侧不同,黄色和绿色)和相反方向的切口位点(Nt。Bvv CI,棕褐色)。 USER矢量用 Pa c I和Nt。 Bbv CI消化,以产生8 nt突出端。尿嘧啶在合适位置的DNA片段(右上角)可以如前所述通过PCR产生,或者通过如本文所述的uNCDF合成。将片段和载体与USER TM TM酶混合物(切除脱氧尿苷,粉红色)和经消化的USER兼容性载体混合。短暂温育后,杂交产物可用于转化E.无需预先结扎。这个数字已经得到了(Nour-Eldin等人,2006年)的允许。

    1. 每个片段将被USER尾部包围,以便插入到USER兼容的向量中。在这个例子中,我们使用我们的 X。 laevis表达载体pNB1u(Nour-Eldin等人,2006)。将uNCDF直接与消化的pNB1u载体混合,而无需事先PCR扩增(图1)。
      1. 在H 2 O(或TE缓冲液pH 8)中将每种uNCDF稀释至100ng /μl。
      2. 将用户兼容的pBlox X.laevis卵母细胞表达载体用PacI / Nt.Bbv C1消化过夜,PCR纯化并稀释至浓度约为50ng /μl(如前所述[Nour-Eldin等人,2006和2010])。
        注意:如果用户兼容的矢量不可用,可以通过PCR生成它们。在这种情况下,通过USER融合将载体骨架处理为与感兴趣的DNA片段组装的DNA片段(请参见下文)。
      3. 对于USER反应,将100ng uNCDF与50ng消化的pNB1u,1U USER酶(NEB),2μl5x PCR反应缓冲液和5μlH 2 O混合。
      4. 在37℃孵育反应25分钟,然后在室温下孵育25分钟。
      5. 转化50微升化学能力的E。通过热休克(冰上5分钟,42℃下30-45秒,冰上5分钟)与反应混合物混合。
      6. 将转化混合物铺在含有适当抗生素选择的LB平板上(对于pNB1u,我们使用羧苄青霉素或含氨苄青霉素的LB平板)。
      7. 从平板上选择三个菌落,过夜培养。提取质粒并通过凝胶电泳分析提取的质粒。用插入序列对质粒进行测序。

  3. 设计和订购含有尿嘧啶的NCDF用于多个片段的用户融合
    为了将多个uNCDF融合到USER兼容的载体中,必须选择重叠区域并将其包括在相邻片段中。如先前所述通过在底链上发现T以及在第一T的下游7-15个碱基处的顶链上的T来选择重叠区域。选择的Ts对应在距接合位点20-30个碱基的距离内图2)(Geu-Flores等人,2007; Nour-Eldin等人,2010)。这些Ts将在合成过程中被U取代。为了插入用户兼容的矢量,我们的通用5'和3'用户尾部被添加到终端片段。当命令合成的DNA时,我们通常密码子优化其中基因将被表达的生物体的序列。在下面的例子中,我们展示了如何设计和排序三个片段进行融合,并通过USER克隆插入USER兼容向量。全长序列(〜7,200bp)被分成三个片段(FAS1_A,FAS1_B和FAS1_C)。它们的终端序列如下。在每个片段中,中间的标点符号表示大约2kb的序列,该序列包括在内,但为了简洁起见在此省略。每个片段被设计为包括与接合点附近的相邻片段的重叠区域。有关如何设计USER融合重叠区域的更多详细信息,请参见下文(Geu-Flores等人,2007; Nour-Eldin等人,2010) 。或者,Amuser网络服务器工具可以为任何USER融合方法设计重叠区域(Genee等人,2015年)。根据Geneart的指示,我们向他们提供了一个包含要在FASTA格式中合成的序列的Word文件。在Word文档的开始部分,我们包含了合成公司的一般说明,下面用斜体标出。
    亲爱的合成公司:
    绿色标志T必须替换为U.绿松石标志着一个A,它与另一个T上的T互补,必须被U取代(即它是T上的T必须由尿嘧啶取代的相反的链)。任何颜色阴影序列都不允许改变。未标记的序列代表编码序列,其可以被改变以克服合成挑战。基因将在非洲爪蟾卵母细胞中表达。请使用适当的密码子偏好表进行密码子优化和改变。



    注意:只有有义链包含在排序过程中,但结果是含有尿嘧啶的双链片段。在密码子优化过程中,任何有色区域都不允许改变。黄色标志着我们的标准5'用户尾巴。绿色标记T,必须合成为U.绿松石标记A,其互补的T必须合成为U.灰色标记重叠,将在USER处理时以单链片段形式暴露,并且将与互补的单个在相邻的片段(在这种情况下是FAS1_B)上搁浅悬垂。合成后,双链FAS1_A uNCDF将如下所示:



    用USER酶处理后,双链FAS1_A uNCDF会丢失位于尿嘧啶上游的序列,如下所示:



    至于FAS1_A,只有有义链包含在订购过程中。绿色标记T,必须合成为U.绿松石标记A,其互补的T必须合成为U.灰色标记将在USER处理时作为单链突出体暴露的重叠,并且其将与互补的单链杂交(在这种情况下,左侧的“灰色”将与FAS1_A上产生的突出端杂交,而右侧的“灰色”将与FAS1_C上产生的突出端杂交)。一旦合成,双链FAS1_B uNCDF将如下所示:



    在用USER酶处理后,双链FAS1_BuNCDF会丢失位于尿嘧啶上游的序列,如下所示:



    至于FAS1_A,只有有义链包含在订购过程中。绿色标记T,必须合成为U.绿松石标记A,其互补T必须合成为U.灰色标记重叠,在USER处理时将作为单链悬垂暴露,并且将与单倍体杂交(在这种情况下,FAS1_B上最右边的“灰色”重叠区域)。一旦合成,双链FAS1_B uNCDF将如下所示:



    用USER酶处理后,双链FAS1_CuNCDF会丢失位于尿嘧啶上游的序列,如下所示:



  4. 将多个uNCDF融合并克隆到线性化的USER兼容向量中


    图2. USER融合技术概述。碎片X1,X2和X3将被融合在一起。重叠区域标记为灰色。尿嘧啶可以通过PCR或如本文所述在合成过程中插入。将DNA片段与预消化的USER兼容性载体混合,并用脱氧尿苷切除的USERTM酶混合物处理。这产生了3个互补的互补序列(箭头所示),而最外侧的序列与预消化载体的突出端互补。这种设计使得能够形成可以直接转化成E的稳定的环状杂交产物。无需预先结扎。这个数字已经得到了(Geu-Flores等人,2007年)的许可。

    为了将多个uNCDF融合并克隆到由我们实验室生成的USER兼容的载体中,我们将通用的8bp长的5'和3'用户尾部添加到末端片段的末端。在交界处包含尿嘧啶的重叠部分被包括在内以允许混合时的无缝融合。在可能的情况下,我们努力选择不同长度和组成的融合重叠,以尽量减少片段之间的杂交。在这个例子中,我们将三个片段融合并克隆到我们的 X中。 laevis表达载体pNB1u(Nour-Eldin等人,2006)。每个片段在每个USER尾部的适当位置包含尿嘧啶。尿嘧啶在合成期间被并入。因此,uNCDF以等摩尔比率直接与消化的pNB1u载体混合,而不用含尿嘧啶的引物进行预先的PCR扩增。
    1. 在H 2 O中稀释每个uNCDF至50ng /μl。
    2. 与用户兼容的pNB1u X。卵母细胞表达载体用PacI / Nt.BbvC1消化过夜,PCR纯化并稀释至约50ng /μl的浓度(如前所述(Nour-Eldin等人,2006和2010))。
    3. 对于USER反应,将100ng每种uNCDF与50ng消化的pNB1u,1U USER酶(NEB),2μl5x PCR反应缓冲液和H 2 O混合以使总体积达到10μl 。
    4. 在37℃孵育反应25分钟,然后在室温下孵育25分钟。
    5. 转化50微升化学能力的E。用反应混合物(冰上5分钟,42℃30分钟-45秒,冰上5分钟)洗涤大肠杆菌(例如热休克的NEB10β)。
      注意:通常添加的USER反应混合物的体积不应超过感受态大肠杆菌体积的10%(即最大5μl反应混合物至50μl大肠杆菌体积,10μl反应混合物至100μlE.大肠杆菌体积等。超过这个比例可以降低转化效率。
    6. 将转化混合物铺在含有适当抗生素选择的LB平板上(对于pNB1u,我们使用含有羧苄青霉素的LB平板)。
    7. 从平板上挑选8个菌落,过夜培养。提取质粒并通过凝胶电泳分析提取的质粒。用插入序列对质粒进行测序。

笔记

如果在第一次试验中插入不成功,我们鼓励用户(除了USER克隆)将uNCDF克隆到钝端克隆载体(例如pJET)中以保存和将来工作。

致谢

MEJ得到了丹麦独立研究委员会(DFF-6108-00122)的资助。 DX,AP,CW,DV,SKL和HHN由丹麦国家研究基金会的DNRF99资助。 ZNB由丹麦创新基金会资助。序号:76-2014-3,西北地区由人类前沿科学计划RGY0075 / 2015资助。作者声明不存在利益冲突或利益冲突。

参考

  1. Cavaleiro,A.M.,Kim,S.H。,Seppala,S.,Nielsen,M.T。和Norholm,M.H。(2015)。 通过优化的尿嘧啶切除克隆实现准确的DNA装配和基因组工程 ACS Synth生物学4(9):1042-1046。
  2. Genee,H.J.,Bonde,M.T.,Bagger,F.O.,Jespersen,J.B.,Sommer,M.O.,Wernersson,R.和Olsen,L.R。(2015)。 软件支持的用于定点诱变和DNA装配的USER克隆策略 ACS Synth Biol 4(3):342-349。
  3. Geu-Flores,F.,Nour-Eldin,H.H.,Nielsen,M.T。和Halkier,B.A。(2007)。 用户融合:快速高效的同时融合和克隆多种PCR产物的方法 Nucleic Acid Res 35(7):e55。
  4. Jorgensen,M. E.,Xu,D.,Crocoll,C.,Ramirez,D.,Motawia,M. S.,Olsen,C. E.,Nour-Eldin,H. H.和Halkier,B. A.(2017)。 NPF家族中转运蛋白底物特异性的起源和进化 Elife < / em> 6.
  5. Nour-Eldin,H. H.,Geu-Flores,F.和Halkier,B. A.(2010)。 用户克隆和用户融合:小型和大型实验室理想的克隆技术 <方法Mol Biol 643:185-200。
  6. Nour-Eldin,H.H.,Hansen,B.G.,Norholm,M.H.,Jensen,J.K。和Halkier,B.A。(2006)。 推进基于尿嘧啶切除的克隆技术,克隆PCR片段的理想技术 核酸研究34(18):e122。
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Copyright Jørgensen et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Jørgensen, M. E., Wulff, N., Nafisi, M., Xu, D., Wang, C., Lambertz, S. K., Belew, Z. M. and Nour-Eldin, H. H. (2017). Design and Direct Assembly of Synthesized Uracil-containing Non-clonal DNA Fragments into Vectors by USERTM Cloning. Bio-protocol 7(22): e2615. DOI: 10.21769/BioProtoc.2615.
  2. Jorgensen, M. E., Xu, D., Crocoll, C., Ramirez, D., Motawia, M. S., Olsen, C. E., Nour-Eldin, H. H. and Halkier, B. A. (2017). Origin and evolution of transporter substrate specificity within the NPF family. Elife 6.
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