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. 2021 Apr 7;24(5):102400. doi: 10.1016/j.isci.2021.102400

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© 2021 The Author(s)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Characterization of the CcpA regulation in vivo for trehalose-inducible system

(A) The fluorescence intensity and OD 600 were measured for the strain carrying pBLTE plasmid under ten conditions (control: 1.5% trehalose, 1.5% trehalose + 1.5% glucose, 1.5% trehalose + 1.5% fructose, 1.5% trehalose + 1.5% mannitol, 1.5% trehalose + 1.5% glycerol, 1.5% trehalose + 1.5% glycerol, 1.5% trehalose + 1.5% saccharose, 1.5% trehalose + 1.5% mannose, 1.5% trehalose + 1.5% sorbitol, 1.5% trehalose + 1.5% arabinose, 1.5% trehalose + 1.5% xylose). Data are shown as means ± SD, n = 3.

(B) Quantify the CCR/CCA effect with the formula I=(FI₁-FI₂)/FI₁×100% due to different carbohydrates (FI₁ represents the fluorescence intensity when only trehalose is added. FI₂ represents the fluorescence intensity when trehalose is added and another carbohydrate is also added. Glucose, fructose, mannitol, glycerol, sucrose, mannose, sorbitol, arabinose, and xylose added at a concentration of 1.5% while adding 1.5% trehalose. Data are shown as means ± SD, n = 3.

(C) The fluorescence intensity and OD 600 were measured for trehalose promoter in both the strain BlspTE (control 1: 1.5% trehalose, 1.5% trehalose + 1.5% glucose, 1.5% trehalose + 1.5% glycerol) and CcpA-deletion strain BlspTE1 (control 2: 1.5% trehalose, 1.5% trehalose + 1.5% glucose, 1.5% trehalose + 1.5% glycerol). Data are shown as means ± SD, n = 3.

(D) The fluorescence intensity and OD 600 were measured for trehalose promoter whose CRE site was replaced by CRE_Tre site in both the strain BlspT1E (control 3: 1.5% trehalose, 1.5% trehalose + 1.5% glucose, 1.5% trehalose + 1.5% glycerol) and CcpA-deletion strain BlspT1E1 (control 4: 1.5% trehalose, 1.5% trehalose + 1.5% glucose, 1.5% trehalose + 1.5% glycerol). Data are shown as means ± SD, n = 3.

(E) The fluorescence intensity and OD 600 were measured for trehalose promoter whose CRE site was replaced by CRE_Tre(R) site in both the strain BlspT2E (control 5: 1.5% trehalose, 1.5% trehalose + 1.5% glucose, 1.5% trehalose + 1.5% glycerol) and CcpA-deletion strain BlspT2E1 (control 6: 1.5% trehalose, 1.5% trehalose + 1.5% glucose, 1.5% trehalose + 1.5% glycerol). Data are shown as means ± SD, n = 3.

(F) Compared three CRE sites by using formula I=(FI₁-FI₂)/FI₁×100% while extra adding glucose or glycerol. Data are shown as means ± SD, n = 3.