Part:BBa_K3805886

OmpT-mCherry

This composite part consists of two parts: OmpT signal peptide (BBa_K3805532)[1]and mCherry (BBa_J06504)[2].Outer membrane protease T(OmpT) is a 33.5 kDa endoprotease[1] located on the outer membrane of Escherichia coli. Ompt signal peptide is an excretion tag linked on the N-terminus of proteins. It can transport proteins across the outer membrane with the help of OmpT. If Ompt signal peptide is fused with mCherry, it will enable mCherry to be secreted to extracellular space[2].

Usage and Biology

Designed together with modified PmrB-anti-mCherry receptor that specifically binds to mCherry(BBa_K3805238)[3], this part acts as a two component signal transduction system whose density can be monitored by fluorescence intensity. The OmpT signal peptide in this part offers a practical and universal strategy for recombinant protein extracellular secretion in Escherichia coli.

Improvement

We fuse OmpT signal peptide at the N-terminus of mCherry to transport mCherry across outer membrane. MCherry itself cannot be secreted to the extracellular space. After the insertion of the OmpT signal peptide, mCherry is able to secrete out successfully.

Design

MCherry is a red fluorescent protein widely used in biotechnology as a tracer, including molecular labeling and localization of cell components. Due to its unique color and single molecule photostability [1], we adopted mCherry to accomplish our project. Functions of mCherry in our project are as follow: 1.Acts as reporter of the P2 promoter. MCherry is inserted at the downstream of P2 promoter, which can be activated by AIP, therefore we can determine whether P2 promoter is activated by observing the red fluorescence. 2.Acts as signal molecule that induces the cheater’s death. MCherry serves as the ligand that specifically binds to the receptor PmrB-anti-mCherry on the cheater’s cytomembrane and initiates downstream extermination pathways in cheaters, eventually leading to their death. MCherry itself, however, cannot be secreted to extracellular environment, which means that we need to modify mCherry to achieve our goals. Consequently, we insert OmpT signal peptide at the N-terminus of mCherry to help mCherry traverse the outer membrane[3].

Results

Figure 1 Images under fluorescence microscope.(A)mCherry without OmpT.(B)mCherry with OmpT.(Magnification:10×100).

Figure 2 The data of fluorescent density by microplate reader.The orange describes the mCherry intensity without OmpT, and the blue describes the mCherry intensity with OmpT.

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]