Part:BBa_K1031520

Pg-B0031-sfGFP-Terminator (HpaR)

Structure

The structure of Pr and Pg promoters are complex, for they are adjacent and many transcription factors besides HpaR including IHF and CRP participate. Pr and Pg are in the opposite direction. The two binding sites for HpaR, OPR1 and OPR2, perform obvious synergistic effect, making the two operons, hpaR and hpaGEDFHI coordinate with each other on transcription level (Fig 1)

Fig 1 Structure of the intergenic region between hpaR and hpaG (Galán, B. et al, 2003). The elements of the promoters are enclosed by square. Especially, two OPRs responsible for HpaR binding are marked. The transcription direction of hpaR and hpaG are indicated with arrows. The IHF and CRP sites are marked respectively.

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 tunning

Pc promoter J23106 is selected to initiate the transcription of hpaR. Based on this circuit, we constructed a library of RBS (Ribosome Binding Site) for tunning for expression level of reporter gene sfGFP (Fig 2). K1031520 is composed of three elements, Pg promoter regulated by HpaR, RBS B0031[1] and reporter gene sfGFP. However, we haven`t got the obvious induction ratio. It is hypothesized that several overall-controlling sites are located in the promoter. Bacteria will control strictly the expression of the relative genes in rich condition.

Fig 2 Construction of reporter circuit. The orange arrow represents Pg promoter. The green oval stands for RBS B0031. sfGFP coding sequence is shown with dark blue, while terminator B0015[2] is in dark red.