DNA sequence manipulation is essential to molecular biology research and biotechnology. In-Fusion cloning is a potent and adaptable technology for seamless DNA fragment assembly without restriction enzymes or DNA ligases. This method uses DNA polymerases to create complementary overhangs on DNA fragments for accurate and directional assembly. A new fusion tag, carbohydrate-binding module 66 (CBM66) was used to produce Thyroid-like peroxidase (BbePox-1) in E. coli . This survey examined heterologous expression of Branchiostoma belcheri BbePox-1 in E. coli (BL21 DE3). The enzyme's nucleotide sequence is engineered into the pCBM66 expression vector via In-Fusion cloning. This work shows that both sets of primers for the In-Fusion method of cloning isolated Bbe-POX1 and linearized pCBM66. Finally, the expression vector was created. PCR using specific primers to the targeted gene and restriction enzyme use on the vector validated the Bbe-POX1 gene's insertion into the pCBM66 vector.
This research was supported by the Slovak Research and Development Agency grant APVV-21-0227.
[1] Park J., Throop AL, LaBaer J. (2015) CP mol. Biol. Vol. 110(1), p. 3.
[2] Fu C., Donovan WP, Shikapwashya-Hasser O., Ye X., Cole RH (2014) PLoS ONE. 9(12), p. 5.
[3] Yu K., Liu C., Kim BG, Lee DY (2015) Biotechnol. Adv. Vol. 33(1), p. 155.
[4] Hamid M., Rehman K. (2009) Food Chem. Vol. 115(4), p. 1177.
[5] Cuskin F, Flint NPP, Gloster TM, Morland C, Basle A, Henrissat B, Coutinho PM, Strazzulli A, Solovyova A, Davies GJ, Gilbert HJ (2012) Proc. Natl. Acad. Sci. Vol. 109(51), p. 20889.
[6] Wanker E., Huber A., Schwab H. (1995) App.l Environ. Microbiol. Vol. 61(5) p. 1953.
[7] Ko H., Kang M., Kim MJ, Yi J., Kang J., Bae JH, Sohn JH (2021) Microb. Cell. Fact. Vol. 20(1), p. 232.