Part:BBa_K4055525

pT7-PhlO

An modified T7 phage promoter which can be regulated by the repressor PhlF

In order to reduce the amount of leakage expression, referring to the structure of BL21 (DE3) +pET28 protein expression system, we hoped to make the T7 promoter and T7RNAP controlled by PhlF at the same time to reduce the leakage activity, so we added PhlO sequence after the T7 promoter.

Experience

BBa_K3254024 element was connected downstream of both the original T7 promoter and the modified T7 promoter with PhlO binding site, which was used to characterize the strength of the promoter.

Schematic of promoter binding

BBa_K2525007 connected to the pSB1K3 skeleton was transferred into the system, and 1mM IPTG induction was added to make it express sufficient PhlF. We took 200 microl cultures into 96-well plates and measured 485/510 fluorescence and OD600 using a microplate reader. The expression level of sfGFP was expressed by the ratio of fluorescence value to OD600. The results showed that for PT7-PHLO, PhlF could significantly inhibit its activity, up to 70 times.

PhlO and PhlF assay results

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
  COMPATIBLE WITH RFC[1000]

Reference

Zong, Y., Zhang, H. M., Lyu, C., Ji, X., Hou, J., Guo, X., Ouyang, Q., and Lou, C. (2017) Insulated transcriptional elements enable precise design of genetic circuits, Nature communications 8, 52.