Part:BBa_K496004
Reporter of Salmonella secretion signal with NLS, GFP, etc
We have made a T3SS test construct on the tetracycline resistant plasmid [pSB1T3] and transform it to the E. coli (K-12) carrying pBAC-SPI-2 as shown in (Fig. 1). It was reported that N-terminus 191 amino acid residues of SlrP (one of the natural effector protein of SPI-2 encoded T3SS) function as a T3 secretion signal domain in Salmonella [2]. And it was also reported that GFP can be secreted through T3SS of Yersinia [1]. So, we fused the N-terminus 191 a.a. of SlrP with the N-terminal of GFP. In addition we located triple NLS (Nuclear Localization Signal) repeats between the T3 secretion signal and GFP so that the injected GFP would localize inside of the nucleus in the target cell. This [T3signal-NLS-GFP] fusion gene is under control of inducible arabinose promoter.
It is possible to join RFP reporter gene down stream of the to GFP construct. RFP reporter can be under control of a constitutive promoter. So, if arabinose is added to the culture medium, E. coli produces both GFP and RFP so that the bacteria become yellow. However, if arabinose is not added, only RFP is produced so that the bacteria become red. We have registered the T3SS signal domain mentioned above as a new BioBrick part.
From results, NLS didn't work correctly so the injected protein was only localized in cytosol but didn't go further to nucleus as intended.
Please check the secretion tag used in this system BBa_K496003.
Fig. 2 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
- 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.
- 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2517
Illegal SapI site found at 961
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