Abstract
Objective
Concatenation of two NdeI–XhoI gene fragments via an oligonucleotide linker on a plasmid vector with an SfiI site was performed to evaluate success rates in construction of polycistronic genes expressible in Escherichia coli.
Results
A series of plasmids with an SfiI site between the selection marker and the replication origin were constructed. The three wheat eEF1B subunit genes inserted between the NdeI and XhoI sites of pET-22b were transferred to the SfiI-containing plasmid with a spectinomycin-resistance gene. Then, the marker gene in the resultant plasmids was substituted with the ampicillin-resistance gene. These plasmids were used for concatenation of two different genes via a linker oligonucleotide containing a ribosome-binding site. During these operations, 42 clones were picked up out of which 41 had the intended product plasmid.
Conclusion
This method, named as the SfiNX method, is useful for trial-and-error based testing of different combinations of fusion and co-expression partners for optimization of recombinant protein production.
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Acknowledgments
We thank Drs. Yasunari Ogihara and Bernd Bukau for providing us plasmids through the Natinal BioResource Project Wheat and Addgene, respectively. The work is supported in part by JSPS KAKENHI Grant Numbers 23310141 and 26640129. KH prepared the pET-22b plasmids with the eEF1B subunit inserts and initiated the idea that every ORF should be shaped in the same format as in this work. KT did everything except for the works of KH.
Supporting information
Supplementary materials and methods.
Supplementary Table 1 PCR primers used in this study.
Supplementary Table 2 Plasmids constructed by insertion of short linkers between the NdeI and XhoI sites.
Supplementary Table 3 Examples of parts that can be prepared from the plasmids created in this study.
Supplementary Fig. 1 A schematic drawing of the procedure for construction of pAET1.
Supplementary Fig. 2 Construction of pAECT1 and pAECT2.
Supplementary Fig. 3 Schematic drawings of plasmids with p15A and pSC101 replication origins constructed in the present study.
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Takai, K., Hisamatsu, K. SfiNX: a method for assembly of protein coding sequences with high success rates. Biotechnol Lett 38, 773–778 (2016). https://doi.org/10.1007/s10529-016-2042-2
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DOI: https://doi.org/10.1007/s10529-016-2042-2