Team:NKU CHINA/Demonstrate
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Demonstrate
3. Population Quality Control (PopQC) System improving the yield of poly-γ-glutamate (γ-PGA)
The original intention of our project is to improve the yield of target metabolite using the PopQC system. We took γ-PGA as an example and measured the γ-PGA yield in both the previous constructed B. amyloliquefaciens NK-Ipop, which contained the pHT01-mCherry-LacI-PgltAB-Pgrac-TetA-GFPint plasmid, and the wild type strain. It can be seen that γ-PGA yield of the NK-Ipop was significantly higher than that of the wild strain. (Fig. 1)
Fig. 1 The γ-PGA yield of B. amyloliquefaciens NK-Ipop and LL3 Δbam. B. amyloliquefaciens NK-Ipop was cultured in the fermentation medium with 10 μg/mL tetracycline for 24 hours. and the wild strain was cultured in the fermentation medium without antibiotics. Data indicate mean values ± standard deviations from three independent experiments performed for B. amyloliquefaciens NK-Ipop, 2 independent experiments performed for B. amyloliquefaciens LL3 Δbam.
4. Safety
Taking safety into consideration, we must ensure that none of our microbe can grow outside of lab in case of leaking out. We verified our Asp auxotroph in B. amyloliquefaciens LL3 which was constructed by a markerless gene replacement method in order to delete its original pathways of synthesizing Asp. We provided the same medium without Asp to both Asp auxotroph and wild type of B. amyloliquefaciens LL3, the result indicated that Asp auxotroph couldn’t grow in Asp-free medium while the wild type cangrew normally.
Fig. 4 Asp auxotroph mutant's growth condition in the medium with or without Asp. The Asp auxotroph mutant was cultured in M9 medium with or without asparagine, separately. 2 independent experiments were carried out.
3. Population Quality Control (PopQC) System improving the yield of poly-γ-glutamate (γ-PGA)
The original intention of our project is to improve the yield of target metabolite using the PopQC system. We took γ-PGA as an example and measured the γ-PGA yield in both the previous constructed B. amyloliquefaciens NK-Ipop, which contained the pHT01-mCherry-LacI-PgltAB-Pgrac-TetA-GFPint plasmid, and the wild type strain. It can be seen that γ-PGA yield of the NK-Ipop was significantly higher than that of the wild strain. (Fig. 1)
Fig. 1 The γ-PGA yield of B. amyloliquefaciens NK-Ipop and LL3 Δbam. B. amyloliquefaciens NK-Ipop was cultured in the fermentation medium with 10 μg/mL tetracycline for 24 hours. and the wild strain was cultured in the fermentation medium without antibiotics. Data indicate mean values ± standard deviations from three independent experiments performed for B. amyloliquefaciens NK-Ipop, 2 independent experiments performed for B. amyloliquefaciens LL3 Δbam.
4. Safety
Taking safety into consideration, we must ensure that none of our microbe can grow outside of lab in case of leaking out. We verified our Asp auxotroph in B. amyloliquefaciens LL3 which was constructed by a markerless gene replacement method in order to delete its original pathways of synthesizing Asp. We provided the same medium without Asp to both Asp auxotroph and wild type of B. amyloliquefaciens LL3, the result indicated that Asp auxotroph couldn’t grow in Asp-free medium while the wild type cangrew normally.
Fig. 4 Asp auxotroph mutant's growth condition in the medium with or without Asp. The Asp auxotroph mutant was cultured in M9 medium with or without asparagine, separately. 2 independent experiments were carried out.