Part:BBa_K1031803

Pu-B0031-sfGFP-Terminator (XylR)

For detailed information concerning XylR and Pu promoter, please visit 2013 Peking iGEM Biosensor XylR

Structure

Pu promoter which is activated by XylR, is σ-54 dependent. It is composed of three elements. The UBS (Upstream Binding Site) site which is responsible for interacting with XylR transcriptional factor. The IHF binding site which allows IHF to participate, enhancing transcription efficiency. -24 and -12 region interact with σ-54 factor of RNA polymerase, enabling the formation of open complex. (Fig 1)

Fig 1 Structure of Pu promoter. The UAS of this promoter shown as blue sequence in the blue frame interacts with DmpR. IHF binding site is shown in green in the green frame. The orange sequence indicates σ54 binding site as -24 region and -12 region. The G in red represents +1 site.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 167
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 219

Construction and tunning

Pc promoter J23114 is selected to initiate the transcription of XylR. Based on this circuit, we constructed a library of RBS (Ribosome Binding Site) including B0031[1], B0032[2], B0033[3] and B0034[4] for tunning for expression level of reporter gene sfGFP. K1031803 consists of Pu promoter, RBS B0031 and reporter gene sfGFP (Fig 2a). Induction ratio was calculated from fluorescence intensity, showing the performance of K1031803(Fig 2b)

Fig 2 Construction and tunning of reporter circuit Pu-B0031-sfGFP (a) The orange arrow represents Pu promoter for XylR. The green oval stands for RBS B0031. sfGFP coding sequence is shown with dark blue, while terminator B0015[5] is in dark red. (b) Horizontal axis represents biosensor circuit J23114-XylR adopting different RBS. Vertical axis stands for induction ratio. The dashed box refers to data for reporter circuit Pu-B0031-sfGFP in collocation with biosensor circuit J23114-XylR.