Coding

Part:BBa_K4229037

Designed by: Nikita Edel   Group: iGEM22_Freiburg   (2022-09-29)
Revision as of 18:47, 10 October 2022 by NikitaEdel (Talk | contribs)


T1-Protein from the synthetic wiffleball (derived from HO-shell) fused to SnoopTag and SpyTag

The T1 protein with snoop- and spyTag structure pore struckture in the modified minimal-wiffleball(BBa_K4229047).In the modified full wiffleball (BBa_K4229047) it is one of the two pore structures, the other one is built by the T2-(BBa_K4229035) and T3-protein(BBa_K4229036).In the minimal as well as in the full wiffleball T1 builds pseudohexamers. This is the modified version of T1, which allows to recruit proteins inside the shell.

A: Showing the original HO-Shell found in Haliangium orchaceum. B: showing the full wiffleball made with H-/T1-/T2-/T3-protein. C: minimalwiffleball made just our of the H- and T1-protein.
























By Western Blotting, the expression of the T1 could be proven by a 29kDa (T1 w/o tags) and a 37kDa (T1) band (Figure 4 and 5). The absence of the Spy/Snp-tag resulted in one single band. When also the tag is expressed, a second band with a shift to around 80kDa was observable. The molecular weight of mVenus2-SpyCatcher has the same size as the T1 with the tags (37kDa). In the full wiffleball, as well in the minimal one, the catching is functioning. Always a small fraction of the unbound T1 was found in the insoluble fraction of the cells, but not when fused to mVenus2, which excludes the possibility that the nature of foci results from insoluble T1-mVenus2-aggregates. Most insoluble T1 was found in the pellet of the minimal Wiffleball, when mVenus2 was also expressed.


Western Blot comparison of the BMC minimal wiffleball with and w/o tags (pHT1) + mVenus2
Western Blot comparison of the BMC full wiffleball with and w/o tags (pT1T2T3) + mVenus2





















The plasmid with this protein was sent kindly send to us by the Kerfeld Group after we found this very interesting paper:

H. Kirst and C. A. Kerfeld, “Bacterial microcompartments: Catalysis-enhancing metabolic modules for next-generation metabolic and biomedical engineering,” BMC Biol., vol. 17, no. 1, pp. 1–11, 2019, DOI: 10.1186/s12915-019-0691-z.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 709
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 36
    Illegal AgeI site found at 513
    Illegal AgeI site found at 621
  • 1000
    COMPATIBLE WITH RFC[1000]


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