Evaluation of directly using Escherichia coli cell lysate expressing Bst DNA polymerase enzyme for Loop-mediated isothermal amplification reaction (LAMP)

Abstract

    Bst DNA polymerase Large Fragment (BstLF) is a crucial enzyme responsible for synthesizing new DNA strands in loop-mediated isothermal amplification (LAMP) reactions. At present, commercially available BstLF for LAMP is costly, whereas self-synthesized BstLF requires a complex purification process and poses challenges in maintaining long-term enzymatic activity. This study aims to simplify the production process, reduce time and costs by expressing BstLF in Escherichia coli BL21(DE3) and directly utilizing the cell lysate in LAMP reactions. BL21(DE3) cells were induced to express BstLF using 0.05 mM IPTG at 37 °C for 4 hours. E. coli BL21(DE3)/BstLF cells were resuspended in Mili-Q water, subjected to heat treatment at 65 °C for 30 minutes, followed by centrifugation to pellet the cells. The cell lysate was directly incorporated into the LAMP reaction. The amplification capability of BstLF was evidenced when lysate from 6 × 10¹¹ cells was included in the reaction, enabling the detection of a minimum of 10³ fg/reaction of genomic DNA from common bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella enterica. This study demonstrates that E. coli BL21(DE3)/BstLF cell lysate can serve as an effective alternative to purified BstLF in LAMP reactions, thereby providing a cost-effective and practical resource for research and educational purposes.