Part:BBa_K4144007

This part express exogenous transporter opPet8p to transport SAMe.

Results

This part is designed to express exogenous transporter opPet8p to transport SAMe. It contains the xylose induced promoter Pxyl, which can be repressed by XylR but can be activated by inducer xylose. SpoVG RBS is a strong ribosome binding site from Bacillus subtilis. 6xHis tag is used to verify the expression of opPet8p. opMistic is derived from Mistic, BBa_K2622006. It was recorded as a unique hydrophilic protein from Bacillus subtilis that works as a fusion tag and helps to integrate other proteins into lipid membranes more easily. Past teams have used an edited Mistic to guide exogenous membrane protein in E. coli. And we plan to optimize such Mistic and used it to guide our transporter opPet8p to bacterial plasma membrane.
When fused to the N-terminus of integral membrane proteins, opMstX enables the cargo proteins to fold into their native conformations in the membrane, thus yielding a high-level expression. With opMistic, opPet8p is supposed to be facilitated to localize into the prokaryotic membrane with higher efficiency. sfGFP is fused to C-terminus to display expression and subcellular location of opPet8p. Pet8p, also known as Mitochondrial SAM carrier (Sam5p), encoded by YNL003c. It is 284 amino acids long and has the characteristic features of the mitochondrial carrier family. Pet8p was overexpressed in bacteria, reconstituted into phospholipid vesicles and identified from its transport properties as a SAM carrier. 
The expression of opPet8p has been verified through visual green fluorescence, Western Blot, and Fluorescent microscopy, we supposed that it might have membrane localization. (Fig. 1).

Figure. 1 Expression verification and membrane localization of SAMe Transport Device

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 356
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 340
  

Reference

[1]Kefala G, Kwiatkowski W, Esquivies L, Maslennikov I, Choe S. Application of Mistic to improving the expression and membrane integration of histidine kinase receptors from Escherichia coli. J Struct Funct Genomics. 2007 Dec;8(4):167-72. doi: 10.1007/s10969-007-9033-4. Epub 2007 Nov 6. PMID: 17985211.