Part:BBa_K496003

Fig. 1

RBS + SlrP secretion tag, salmonella T3 sec. sys.

   Leucine-rich repeat protein (SlrP) is one of the effector protein secreted by Salmonella Type 3 Secretion System(T3SS). It is known that N-terminus 191 amino acid residues can be used as secretion tag^[1]. We made this fragment in a BioBrick part. RBS was added preceeding tag coding region. Between RBS and tag coding sequence there only 4 base pairs. In theory it can be used to secrete tagged proteins through Type 3 Secretion Apparatus(T3SA). Due to functional limitations of T3SA it's known that it can't transport very stable protein domains like zinc fingers. Regardless it was shown to transport such stable proteins as GFP^[2].

   See BBa_K496004 for attempts to use this part to inject GFP to eukaryotic cell.

   This tag was used in BBa_K496004. Also to distinguish GFP in the E.coli and GFP in the target cells constitutive RFP (BBa_J04450) reporter was added down stream of BBa_K496004. The results of this experiment can be seen bellow. The results are encouraging because you can see in Fig. 1 GFP located in the form of target cell.

   Fig. 1 Cells were observed under Confocal Laser Scannning Microscope(OLYMPUS FV-1000D) with blue(473 nm) or green(559 nm) excitation lights at 7.5 hrs after first exposure. When arabinose was added to medium, E.coli have expressed GFP beside constitutively expressed RFP, so [A-4] E. coli cells are indicated by yellow color, and GFP injected into ekaryotic cells are indicated by green color. Compairing [A-1] to [A-4] it can be seen that GFP is located in the cytosol of RK13. In contrast, compairing [B-4] and [C-4] GFP weren't find in the cytosol.

References

1. Miao EA, Miller SI. 2000. A conserved amino acid sequence directing intracellular type III secretion by Salmonella typhimurium. Proc Natl Acad Sci U S A. Vol.97:7539-7544.
2. Jacobi CA, Roggenkamp A, Rakin A, Zumbihl R, Leitritz L, Heesemann J. 1998. In vitro and in vivo expression studies of yopE from Yersinia enterocolitica using the gfp reporter gene. Mol Microbiol. Vol.30:865-882.

1. http://www.ncbi.nlm.nih.gov/nuccore/AE006468

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]