Part:BBa_K3580101:Experience

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Applications of BBa_K3580101

In our project, we inserted this parts into pBbe5a(AmpR colE1-ori high copy plasmid., BglBrick vectors) plasmid.

1. in vivo limonene synthesis

Introduction

Fig. 1-1 Schematic of in vivo monoterpene synthesis

Before monoterpene synthesis with cell-free system, we confirmed activity of the genes by monoterpene production with E.coli In this experiment, DH5-alpha transformed with a plasmid encoding enzymes of the mevalonate pathway (pBbA5c-MevT-MBI from addgene) and a plasmid encoding GPP synthetase and monoterpene synthase (BBa_K3580101 / BBa_K3580102) were used.

Materials and method

DH5-alpha transformed with pBbA5c-MevT-MBI and BBa_K3580101 / BBa_K3580102 were used in this experiment. DH5-alpha was selected as the strain used because it was based on the strain used in the 2019 XJTU-CHINA team's limonene production project. And DH5-alpha transformed with only pBbA5c-MevT-MBI was used as negative control. Strains of transformed DH5-alpha were grown in 2xYT media (16 g/L tryptone, 10 g/L yeast extract, 7 g/L NaCl. These components were purchased from Nakalai tesque) containing 25 ng/µl (Nakalai tesque) of chloramphenicol and 1 % in mass percent concentration of glucose (Wako). After culturing overnight (37℃, 180 rpm, 14 h) on 3 ml scales, 500 µM IPTG (Nacalai tesque) to induce expression and 500 µl dodecane overlay to capture monoterpene were added when OD600 exceeded 1.5. After induction by IPTG, strains were continuously cultured at 30 ℃ and 200 rpm for 2 days (48 h). After 2 days of culture, 300 µl dodecane overlays were collected, mixed with 700 µl ethyl acetate and analyzed by GC/MS. The GC / MS methods used were as described in 3-3-1.

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