Part:BBa_K1031302

Pc-B0032-sfGFP-Terminator (HbpR)

For detailed information concerning HbpR and Pc promoter, please visit 2013 Peking iGEM Biosensor HbpR

Structure

HbpR is the activator of two promoters, Pc and Pd, they are σ-54 dependent. HbpR binds to UAS C-1 and UAS C-2 on Pc, UAS D-1 and D-2 on Pd. The 32-bp space between the centers of UASs C-1 and C-2 is critical for cooperative interactions. However, when the UASs C-1/C-2 are deleted and UASs C-3/C-4 are placed in an appropriate position with respect to the promoter region, the Pc promoter is still inducible with 2-HBP, albeit at a lower level. It shows that the presence of UAS pair C-3/C-4 mediated a higher promoter activity for transcription of hbpR(Fig 1 a, b)

Fig 1 The sequences preceding hbpC and hbpD promoter contains the binding sites for HbpR (UAS). The UASs are boxed in red. (a) HbpR binding sites on Pc promoter, there are 4 UASs responsible for binding with HbpR. (b) HbpR binding sites on Pd promoter, there are 2 UASs for binding with HbpR.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 266

Construction and data

K1031302 is composed of three elements, the inducible promoter Pc, RBS (Ribosome Binding Site) B0032, and reporter gene sfGFP with terminator B0015. (Fig 2)

Fig 2 Construction of reporter circuit. The orange arrow represents Pc promoter for HbpR. The green oval stands for RBS B0032. sfGFP coding sequence is shown with dark blue, while terminator B0015 is in dark red.

We created a library for the RBS of sfGFP, including B0031, B0032 and B0034. Dose response curve were tested at the presence of 2-ABP and 2-HBP respectively. Experiment results showed that for inducers 2-ABP and 2-HBP, RBS B0032 had a superior performance compared with B0031 and B0034(Fig 3 a, b)

Fig 3 (a) Dose response curve of HbpR when exposed to 2-ABP at the concentration of 0.5µM, 1µM, 5µM, 10µM, 50µM, 100µM respectively. The reporter circuit consists of Pc promoter, RBS B0031, and reporter gene sfGFP. The curve with dark orange represents the circuit with B0032. Three lines represent circuit adopting different RBS. Fluorescence intensity of sfGFP is detected and calculated to plot induction ratio. (b) Dose response curve of HbpR when exposed to 2-HBP at the concentration of 0.5µM, 1µM, 5µM, 10µM, 50µM, 100µM respectively.