Abstract
Key message
In-house production of a positive selection cloning vector could be simple, efficient and low cost.
Abstract
DNA cloning technology requires a vector to harbour a gene of interest for multiplication of the gene in bacterial cells. Positive selection vector has become a popular type of cloning vector due to the simplicity and efficiency of the positive selection system. Due to the presence of a toxic gene, propagation of a commercial positive selection vector in common laboratory E. coli strains is infeasible. This study demonstrated a strategy for propagation and in-house production of a commercial positive selection vector, i.e., pJET1.2/blunt cloning vector, at low cost. This was done by insertion of a specially designed DNA fragment (MCS fragment), which can be easily removed later by EcoRV digestion, into the pJET1.2/blunt cloning vector to allow the propagation of the modified plasmid (termed pJET1.2M) in common E. coli strains. Removal of the MCS fragment from the pJET1.2M plasmid produces the pJET1.2/blunt cloning vector ready for gene cloning. The self-made pJET1.2/blunt cloning vector exhibited a cloning efficiency of 100%.
This is a preview of subscription content, log in via an institution to check access.
References
Choi YJ, Wang TT, Lee BH (2002) Positive selection vectors. Crit Rev Biotechnol 22(3):225–244. https://doi.org/10.1080/07388550290789504
Green MR, Sambrook J (2019) Screening bacterial colonies using X-Gal and IPTG: α-complementation. Cold Spring Harb Protoc. https://doi.org/10.1101/pdb.prot101329
Green MR, Sambrook J (2020) Cloning in plasmid vectors: blunt-end cloning. Cold Spring Harb Protoc. https://doi.org/10.1101/pdb.prot101246
Janulaitis A, Petrušyté M, Butkus V (1983) Three sequence-specific endonucleases from Escherichia coli RFL47. FEBS Lett 161(2):213–216. https://doi.org/10.1016/0014-5793(83)81010-2
Julin DA (2018) Blue/White Selection. In: Wells RD, Bond JS, Klinman J, Masters BSS (eds) Molecular life sciences: an encyclopedic reference. Springer, New York
Motohashi K (2019) A novel series of high-efficiency vectors for TA cloning and blunt-end cloning of PCR products. Sci Rep 9(1):6417. https://doi.org/10.1038/s41598-019-42868-6
Padmanabhan S, Banerjee S, Mandi N (2011) Screening of Bacterial recombinants: strategies and preventing false positives. In: Brown GG (ed) Molecular cloning - selected applications in medicine and biology. IntechOpen, London
Ramos AE, Muñoz M, Moreno-Pérez DA, Patarroyo MA (2017) pELMO, an optimised in-house cloning vector. AMB Expr 7(1):26. https://doi.org/10.1186/s13568-017-0324-2
Reece-Hoyes JS, Walhout AJM (2018) Gateway recombinational cloning. Cold Spring Harb Protoc. https://doi.org/10.1101/pdb.top094912
Seetaraman Amritha TM, Mahajan S, Subramaniam K, Chandramohan Y, Dhanasekaran A (2020) Cloning, expression and purification of recombinant dermatopontin in Escherichia coli. PLoS ONE 15(11):e0242798. https://doi.org/10.1371/journal.pone.0242798
Yusuf CYL, Abdullah JO, Shaharuddin NA, Abu Seman I, Abdullah MP (2018a) Characterization of promoter of EgPAL1, a novel PAL gene from the oil palm Elaeis guineensis Jacq. Plant Cell Rep 37(2):265–278. https://doi.org/10.1007/s00299-017-2228-7
Yusuf CYL, Abu Seman I, Ab Shukor NA, Mohd Nor MN, Abdullah MP (2018b) Cloning and analysis of the Eg4CL1 gene and its promoter from oil palm (Elaeis guineensis Jacq.). Sains Malaysiana. 47(8):1709–1723. https://doi.org/10.17576/jsm-2018b-4708-10
Yusuf CYL, Nabilah NS, Taufik NAAM, Seman IA, Abdullah MP (2022) Genome-wide analysis of the CAD gene family reveals two bona fide CAD genes in oil palm. 3 Biotech 12(7):149. https://doi.org/10.1007/s13205-022-03208-0
Zhang YS (2016) Blue-white screening liquid can eliminate false positives in blue-white colony screening. Genet Mol Res. https://doi.org/10.4238/gmr.15027925
Zhang K, Yin X, Shi K, Zhang S, Wang J, Zhao S, Deng H, Zhang C, Wu Z, Li Y, Zhou X, Deng W (2021) A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation. Sci Rep 11(1):10454. https://doi.org/10.1038/s41598-021-89884-z
Acknowledgements
The authors would like to thank the lab staff, especially Natasha Afzan Tamby Husin, Dahyudeen Dahlan and Muhd Shazreen Sobri.
Funding
This study was supported by Fundamental Research Grant Scheme (Reference Code: FRGS/1/2019/STG05/UITM/02/1) from the Malaysian Ministry of Higher Education (MOHE).
Author information
Authors and Affiliations
Contributions
MPA and CYLY conceived and designed the research. ON performed the study. CYLY and MPA prepared the manuscript. All authors read and approved the manuscript.
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Data availability
Online Resource 1-3 are provided as electronic supplementary material.
Ethical approval
This study does not involve any animal or human participants.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Nawawi, O., Abdullah, M.P. & Yusuf, C.Y.L. A strategy for in-house production of a positive selection cloning vector from the commercial pJET1.2/blunt cloning vector at minimal cost. 3 Biotech 12, 216 (2022). https://doi.org/10.1007/s13205-022-03289-x
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s13205-022-03289-x