The pJOE8999_sfp_degQ plasmid is obtained from the Suzhou Silicon based Biotechnology Co., Ltd (China). After the plasmid was transformed into Bacillus subtilis 168, the kanamycin resistant clones were selected for 0.2% mannose-induction. And the modified clones would be verified by colony PCR and sequencing. And the modified strain would be utilized for fengycins production. After that fengycins would be extracted from fermentation broth and then quantified by HPLC. (Tan et al, 2022)
In this section, composite part BBa_K4274035 was used.
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pW2 plasmid for the expression of ClSS and pW34 plasmid for the expression of ERG20 are obtained from the Suzhou Silicon based Biotechnology Co., Ltd (China). To construct plasmid pW1_CE, pW1_CEM and pW1_TCEM, golden gate assembling and transformation were carried out in steps. Plasmid pW1_CEM_FL is constructed as the following experimental steps: PCR on pW1_CEM plasmid, agarose gel electrophoresis, gel extraction, golden gate assembling and transformation. After verifying by colony PCR and sequencing, pW1_CE, pW1_CEM, pW1_TCEM and pW1_CEM_FL were co-transformed with pMVA into Escherichia coli DH5α, then strain CE, CEM, TCEM, CEM_FL were successfully constructed and utilized for santalene production. After rapid centrifugation, the supernatant of dodecane was spiked with with 0.475 g/L a-humulene as an internal standard, and then injected into GC/MS for quantification analysis of α-santalene production. (Jia Z. et al, 2022)
In this section, composite parts BBa_K4274030, BBa_K4274031, BBa_K4274032 and BBa_K4274033 were used.
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The pJOE8999_patB plasmid is constructed followed by patB_target assembling and homology arms assembling. The short DNA fragment patB_target was obtained by T4 PNK phosphorylation of DNA oligos and then golden gate assembing with pJOE8999 plasmid. Homology arms for repairing were obtained by PCR amplification of Bacillus subtilis and gel extraction. After digestion by Bgl1 and ligation by T4 ligase with pJOE8999_patB_target, the pJOE8999_patB plasmid was successfully constructed.
Same as pJOE8999_sfp_degQ plasmid, patB gene was knocked-out after transformation, induction and elimination. And colony PCR results could be used to prove the clones selected were modified.
In this section, composite part BBa_K4274036 was used.
Visit the protocol below for more details about experimental procedures.