Insertion of Three Genes Encoding Polyhydroxybutyrate into Escherichia coli BL21 as the First Host: A Preliminary Exploration of Plasmid Transformant

Abstract

The use of biodegradable plastics presents a sustainable option as concerns about the environmental effects of petroleum-based plastics grow. PHB (polyhydroxybutyrate) is one of the biodegradable biopolymers, which can be produced by Escherichia coli encoded by the PhaA, PhaB, and PhaC genes. This research aims to determine and study the ability of E. coli BL21 as a host in transforming the PhaA, PhaB, and PhaC genes encoding PHB, and characterizing successful E. coli BL21 carrying each gene. Recombinant plasmids carrying PhaA, PhaB, and PhaC genes were constructed by cloning into a pUC57 vector and transformed into E. coli BL21 by electroporation. The positive transformants were analyzed using colony-PCR, restriction digestion, and Sanger Sequencing to confirm the successful insertion of the target. Recombinant plasmids were isolated, purified, and verified, obtaining target fragments of 1195bp for PhaA, 752bp for PhaB, and 1786bp for PhaC. The results showed that E. coli BL21 bacteria could act as a transformation host for the recombinant plasmid carrying the PhaA, PhaB, and PhaC genes, which was confirmed through growth on ampicillin LB medium. The PhaA, PhaB, and PhaC genes were successfully transformed with recombinant E. coli BL21 and showed a gene size of 1195 bp, 752 bp, and 1786 bp, respectively. Based on sequences analyses showed a high percent identity and query cover against the reference gene database (KP681582, KP681583, and KP681584). The entire PhaA, PhaB, and PhaC gene sequence has been inserted into recombinant cells, with the result of cutting according to the gene band size and the pUC57 plasmid size. This study provides a foundation for the optimal expression of PHB in recombinant bacteria and serves as a reference in designing genetic transformation strategies for E. coli BL21. The method is also of industrial relevance for replacing or reducing conventional plastics, thereby boosting the circular bioeconomy and directly contributing to the UN Sustainable Development Goals, especially in SDG 9 which focuses in industry, innovation, and infrastructure and SDG 12 which focuses in responsible consumption and production.