Part:BBa_K4229037

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

This biobrick code for the T1 protein fused to the SnoopTag and the SpyTag. It is part of a rigid shell bacterial microcompartment called wiffleball [1] . It can be used to either form minimal wiffleballs (BBa_K4229047) or full wiffleballs (BBa_K4229047), the difference being the presence of the T2(BBa_K4229035) and T3 proteins (BBa_K4229036) in the full wiffleball. Both minimal and full wiffleball need also the H protein (present in both biobricks). The T1 protein forms pseudo hexamers. This biobrick describes a modified version of T1, which allows to recruit proteins inside the shell.

[Fig.1]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 29 kDa band (T1 w/o tags) and a 37 kDa band (T1 with tags) (Figures 2 and 3). The absence of the Spy/Snp-tag resulted in one single band. When also the tag is expressed, a second band corresponding to a protein of around 80 kDa was observable. The molecular weight of mVenus2-SpyCatcher is the same of that of the T1 protein with the tags (37 kDa). We could show that proteins can be ligated onto the T1 proteins via the SpyCatcher/SpyTag reaction. A small fraction of the unbound T1 was found in the insoluble fraction of the cells, but not when fused to mVenus2. Most insoluble T1 was found in the pellet of cells expressing the minimal wffleball, when mVenus2 was also expressed.

[Fig.2]Western Blot comparison of the BMC minimal wiffleball with and w/o tags (pHT1) + mVenus2

[Fig.3]Western Blot comparison of the BMC full wiffleball with and w/o tags (pT1T2T3) + mVenus2

The plasmid coding fo the T1 protein was a kind gift by Cheryl Kerfeld.

[1]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]