Part:BBa_K2232022:Experience

iGEM2017 SZU-China

We add the RBS of Bacillus subtilis and a segment of signal peptide at the upstream of cds of Carboxysomal Carbonic Anhydrase(csoS3) from Halothiobacillus neapolitanus, in order to achieve the expression and secretion of this part in the B.subtilis WB800. The construction of the shuttle expression-secretion vector_CsoS3 is shown as follow in figure 1.

Fig.1 Construction of the shuttle expression-secretion vector_CsoS3.

The new part (BBa_K2232022) was constructed into shuttle expression-secretion vector pP43NMK, and 1% Agarose Gel Electrophoresis of the plasmid verified the correct construction in figure 2.

Fig.2 1% Agarose Gel Electrophoresis of Vector_ CsoS3 and its identification by restriction digestion. Lane 1: Complete plasmid; Lane 2: Plasmid digested by KpnI and HindIII; Lane M: DL marker.The length of part CsoS3 was 1774 bp and the blank vector was 6785 bp.

The crude enzyme solution was obtained from the supernatant of fermentation broth of the Bacillus subtilis including the original strain WB800 and the recombinant WB800_ CsoS3. Since the fact that CA can also catalyze the hydration reaction of ester, which can be used to assay the activity of CA, we conducted measurement on CsoS3 according to a method from Verpoorte JA(1967), and the result(Fig.3) certified that the CsoS3 has high esterase activity.

Fig.3 The esterase activity of crude enzyme solution from the supernatant of fermentation broth of the Bacillus subtilis including the original strain WB800 and the recombinant WB800_ CsoS3.

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