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Part:BBa_K1992003

Designed by: Shilo Ohayon   Group: iGEM16_Technion_Israel   (2016-10-08)
Revision as of 14:27, 18 October 2016 by Shilo (Talk | contribs) (→‎Experiments and results)


Tar chemoreceptor tagged with GFP

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1282
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2324
    Illegal SapI.rc site found at 111


Usage and Biology

E.coli native chemoreceptors cluster in the cell poles. This property is critical for signal amplification and adaptation of the cell. Although little is known about the mechanism of localization, it is important to preserve this property with our designed receptors in order to keep a functional and sensitive chemotaxis response (1).

GFP labeling is a very common way to examine the migration and localization of certain proteins in vivo. Fusion of GFP to Tar chemoreceptor enabled us to track the migration and localization of the protein to the cell poles as expected. This part is a composed from three iGEM registry BioBricks.

Design considerations

The Tar receptor (BBa_K777000) and GFP(BBa_B0040) were taken and amplified from the kit. The flexible linker (J18921) was added by using reverse transcription PCR. The C-terminos of Tar

Experiments and results

Fig1. (a) GFP expression in the cytoplam - positive control (b) Tar expression in UU1250 strain cloned with K1992004 expression system - negative control. (c) Tar-GFP expression in UU1250 strain cloned with K1992009 expression system. (d) Tar-GFP expression in UU1250 strain cloned with K1992008 expression system.

The fused Tar-GFP protein was cloned to our expression systems (BBa_K1992008 and BBa_K1992009) in order to examine the migration of the receptor. By using a fluorescence microscope it can be seen in figure 1 that the receptor is closter in the poles of the bacteria as expected.



Reference

1.SHIOMI, Daisuke, et al. Helical distribution of the bacterial chemoreceptor via colocalization with the Sec protein translocation machinery. Molecular microbiology, 2006, 60.4: 894-906.‏


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