Liu et al. BMC Biotechnology (2018) ODOLOGY ARTICLEOpen AccesspXST, a novel vector for TA cloningand blunt-end cloningQin Liu1, Hui-Jie Dang1, Yuan-Hang Wu1, Min Li1, Yin-Hua Chen1, Xiao-Lei Niu1, Kai-Mian Li2 and Li-Juan Luo1*AbstractBackground: With the rapid development of sequencing technologies, increasing amount of genomic informationhas been accumulated. To clone genes for further functional studies in large scale, a cheap, fast and efficient cloningvector is desired.Results: A bifunctional vector pXST has been constructed. The pXST vector harbors a XcmI-ccdB-XcmI cassette andrestriction site SmaI. Digestion the vector with XcmI generates a single thymidine (T) overhang at 3′ end whichfacilitates TA cloning, and SmaI gives blunt end that enables the blunt-end ligation. Multiple products with varioussizes were amplified from cassava genome by PCR and each PCR fragment was separately cloned into a pXST usingTA cloning and blunt-end ligation methods. In general, the TA cloning gave higher transformation efficiency thanblunt-end ligation for inserts with all different sizes, and the transformation efficiency significantly decreased withincreasing size of inserts. The highest transformation efficiency (8.6 106 transformants/μg) was achieved when cloning517 bp DNA fragment using TA cloning. No significant difference observed in the positive cloning efficiency betweentwo ligation methods and the positive cloning efficiency could reach as high as 100% especially for small inserts (e.g.517 and 957 base pairs).Conclusions: We describe a simple and general method to construct a novel pXST vector. We confirm the feasibilityof using pXST vector to clone PCR products amplified from cassava genome with both TA cloning and blunt-endligation methods. The pXST plasmid has several advantages over many currently available vectors in that (1) itpossesses XcmI-ccdB-XcmI cassette and restriction site SmaI, enabling both TA cloning and blunt-end ligation. (2) itallows direct selection of positive recombinant plasmids in Escherichia coli through disruption of the ccdB gene. (3)it improves positive cloning efficiency by introducing the ccdB gene, reducing the possibility of self-ligation frominsufficient digested plasmids. (4) it could be used by high performance and cost-effective cloning methods.Therefore, this dual function vector would offer flexible alternatives for gene cloning experiments to researchers.Keywords: PCR product, Cloning, T-vector, Blunt-end ligation, High efficiencyBackgroundAmplification and cloning of genes are fundamental techniques in the field of molecular biology. DNA ampliconsby the polymerase chain reaction (PCR), require to be introduced into the plasmids for propagation, preservation,sequencing before transferred into expression vectors forthe following functional characterization. The key step forcloning is to fuse the target DNA fragments into the linearized vectors, and many approaches have been developed* Correspondence: [email protected] Key Laboratory for Sustainable Utilization of Tropical Bioresources,Institute of Tropical Agriculture and Forestry, Hainan University, Haikou570228, ChinaFull list of author information is available at the end of the articleto achieve this goal, including “TA” cloning, “blunt-end”cloning, TOPO, Gateway, etc. [1–5].Generally, the currently available cloning methods can bedivided into two categories, according to the criticalenzymes catalyzed in the system: ligase-free methods andligase-dependent methods. Most ligase-free methodsachieved the DNA recombinant through DNA pairing andhomologous recombination, which requires the wholecorresponding systems, including the specific primers andenzymes, such as recombinase or topoisomerase I, thus,increasing the cost and complexity of the methods [6, 7].Due to the high cost and complicated protocol, hardly cancommercially available ligase-free methods meet therequirement for large scale cloning and be used in most The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication o/1.0/) applies to the data made available in this article, unless otherwise stated.
Liu et al. BMC Biotechnology (2018) 18:44Page 2 of 7laboratories. Comparatively, the ligase-dependent methodsare more widely and robustly used, which includecohesive-end ligation, blunt-end ligation and TA cloning. The cohesive-end ligation links the target DNA fragments (inserts) to plasmids through complementary between the inserts and plasmid vectors, resulting in highrecombinant efficiency. However, chances are high that theinserts contain multiple cloning sites, increasing the difficulties to choose the appropriate restriction enzymes. Theblunt-end cloning walks around such problems by avoidingthe digestion of the insert fragments. However new problems arise for the blunt-end cloning because the blunt endsof vectors could be self-ligated, decreasing the available vectors for ligating inserts. TA cloning seems to solve the problem of self-ligation by using the benefit of Taq polymerase.The Taq polymerase could preferentially add a singleadenosine (dA) to the 3′-end of a PCR product which iscomplimentary to the vectors with a single thymine overhang at 3′-end (T-vector) . Therefore, TA cloning is thesimplest and most efficient approach for cloning of PCRproducts, because the method avoid the requirement ofdigesting the insert fragments and also the self-ligationproblem of vectors.After the transformation of the recombinant plasmids,an efficient selection method is required for the screeningof positive colonies that contain the target inserts. Different strategies, including the white-blue screening, fluorescent protein-based method and introduction of antibioticresistance and lethal genes, have been described [10–13].The control of cell death B (ccdB) gene encodes a potentinhibitor of gyrase that induces DNA breaking and celldeath in the absence of the antidote gene CcdA . Suchunique characteristic facilitates the positive selection ofthe recombinant plasmids, because only when the targetDNA fragments were inserted into vectors that disruptthe ccdB genes, the cells could survive [15, 16].In this study, we have constructed a novel cloning vectorpXST by introducing the XcmI-ccdB-XcmI cassette andrestriction enzyme site SmaI into the plasmid pMD-19 T.The pXST could be used by both TA cloning and blunt endligation, providing more options when selection of cloningmethods. In addition, introduction of ccdB genes allows thedirect selection of positive colonies and also reduces theself-ligation of blunt-end vectors. Furthermore, the systemto catalyze the ligation is cheap and fast. Therefore, thisplasmid is expected to be widely used as a high performance and cost effective cloning vector.MethodsMaterialsPlasmids pMD-19 T (simple) (TaKaRa, Dalian, China) andpDONOR221 (Invitrogen Corp., CA, USA) served as thebackbone and ccdB donor vector, respectively. Escherichiacoli DB3.1 (Invitrogen) was employed to hold the destination vector. E.coli strain DH5α was used for the plasmidtransformation and cloning. The LA Taq polymerase withGC buffer (TaKaRa) and Phusion DNA polymerase(Thermo Fisher, Waltham, MA) was employed to DNAfragment amplification. The PCR products purificationand plasmid extraction kits were bought from Omega(Omega, Doraville, USA). T4 ligase and restriction enzymeXcmI and SmaI were purchased from Thermo Fisher.Primer designAll the primers used in this study were designed by theprogram Oligo7. The detail information of sequences andcorresponding product lengths are listed in Table 1. TwoXcmI restriction sites were created by adding sequence (5’Table 1 Primer sequences used in this studyNamePrimer SequencesProduct size CCACGAGAR4ACTAACACTGCAATTCCACGATThe underlined sequence is the specific restriction site for XcmI and the small letters is the complementary sequence for the ccdB amplification210 (insert)126 (insert)51795715152343
Liu et al. BMC Biotechnology (2018) 18:44CCAATACTTGTATGG 3′) to the 5′ ends of primers togenerate the XcmI-ccdB-XcmI cassette.Obtaining the ccdB geneThe pDONR221 vector was used as a template for theccdB amplification using ccdB-F and ccdB-R primers. ThePCR reaction was performed in 50 μL mixture consistedof 1 ng pDONR221 vector, 0.4 μM of each primer, 0.5 μLTaKaRa LA Taq, 2.5 mM dNTP mixture, 10 LA PCRBuffer I (Mg2 Plus) and an appropriate amount ofddH2O. The thermic profile was set as follows: initialdenaturing step at 95 C, 30 s annealing at 56 Cand 30 sextension step at 72 C, followed by a final extension stepat 72 C for 5 min. The PCR product was then purified byThe E.Z.N.A. Gel Extraction Kit (Omega, cat. no. D2501)following the manufacturer’s instructions.pXST constructionThe purified product was cloned into the pMD19-T (simple) vector to yield the pXST vector and the ligation wasperformed according to the manufacturer’s instruction. Theligation mixture was used to transform competent E. coliDB3.1 cells. After the overnight culture, several clones wereselected to detect the orientation of the inserted ccdBfragment using the ccdB-F and M13R primers. The positiveclone was cultured in liquid LB medium for overnight.Finally, the pXST plasmid was extracted using the PlasmidMini Kit (Omega Bio-Tek, USA). The plasmid was digestedwith SmaI to generate the blunt-end fragments and XcmIto create T-vector.The desired fragments were identified by agarose gel electrophoresis and purified by the Gel Extraction Kit (Omega).The pXST lethality was confirmed by transforming thevector into DB3.1 and DH5α competent cells simultaneously and observing the bacteria growth.pXST transformation efficiency and positive cloningefficiencyThe DNA fragments of different lengths were amplifiedfrom the cassava MeSTP7 genomic sequence (Phytozomeaccession: Manes.03G180400). All fragments were amplified with or without 3′ terminal A by using the LA taq andPhusion DNA polymerase respectively. After the PCRproducts were purified, the ligation reaction was performedin 10 μL volume using 50 ng linearized pXST, 50 ng purified fragment, 5 U T4 DNA ligase, 1 μL 10 T4 buffer and6 μL ddH2O. The mixture was incubated at 22 C for 1 hand then added into 100 μL E. coli DH5α chemically competent cells, followed by incubation on ice for 30 min andheat shock at 42 C for 50 s. All suspended cells were platedon a Luria-Bertani (LB)-agar plate with 100 μg/ml ampicillin. After an overnight culture the colonies were counted tocalculate the transformation efficiency by using nsform.htm) andPage 3 of 7analyzed by colony PCR to examine the cloning efficiency.The mean values and standard deviation (SD) werecalculated from the measurements of three independent experiments.ResultsThe construction of the pXST cloning vectorThe pXST vector was constructed followed the flowchart illustrated in Fig. 1. The XcmI-ccdB-XcmI cassettewas amplified by using the pDONR221 vector as a template. The amplification was catalyzed by the polymeraseTaKaRa LA Taq which could add an extra adenine tothe 3′ end of the PCR fragments. The PCR productswere examined by 1% agarose gel. As expected, and theelectrophoresis showed a clear strap around the size of500 bp. The target band was purified and then introduced into the commercial pMD19-T (simple) vector.The orientation of the insert fragment ccdB gene wasexamined by performing colony PCR using the primerpairs, ccdB-F and M13R. After the colony PCR, theproducts were analyzed by gel electrophoresis (Fig. 2a).There were 6 out of 10 colonies showed a clear band,suggesting that the plasmids showing PCR amplificationcontain the ccdB gene with the expected orientation(pXST) whereas the rest four without amplification havethe ccdB gene in the reverse orientation (pXST-R). ThepXST and pXST-R were verified by sequencing. Toexamine if insertion of ccdB gene in the reverse orientation would also lead to cell lethality, both pXST andpXST-R were transformed into E. coli DB3.1 (ccdB resistant strain) and DH5α, and the transformants weregrew on LB plates with ampicillin selection independently. As showed in Fig. 3, DH5α containing pXST can’tgrow on the LB-Amp plates with or without addingIPTG (Fig. 3b, c), suggesting that ccdB gene can be wellexpressed driven by lac promoter even at basal level(without IPTG induction), and its expression caused thelethality. Whereas, DH5α with pXST-R can grow on theLB-Amp plate with adding IPTG (Fig. 3d), which indicated that the insertion of ccdB in the reverse orientation cannot be expressed, therefore, cannot result inlethality. These results also suggested that the ccdB genefragment in pXST and pXST-R does not contain its ownpromoter, and its expression requires to be the driven oflac promoter. Together, the ccdB gene in the pXST isexpressed under the control of lac promoter and causedcell death in DH5α but not in DB3.1.pXST transformation efficiency regarding PCR productsNext, we continued to examine the feasibility of usingpXST as T-vector and blunt-end vector for conductingboth TA cloning and blunt-end ligation. The plasmidpXST was d