Part:BBa_K4229061

sfGFP with targetpeptide for encapsulins regulated by T7 promoter

Usage: Nanocompartments are found in some bacteria and archea. The encapsulin natively expressed in M. Xanthus is composed of the protein EncA [1][2].In M. Xanthus, the encapsulin is known to encapsulate three different cargo proteins, which play a role in iron storage [2]. Cargo can be targeted to the Encapsulin through a native targeting peptide which binds non-covalently to the EncA. sfGFP fused to the targeting peptide allows for the visualization of Encapsulins in vivo.

Experimental results: BL21 were transformed with pBAD_encA and either pET_sfGFP or pET_sfGFP_TP. A larger culture (50 ml) was grown until OD: 0.6-0.8 at 30 °C and induced with arabinose for the Encapsulins and IPTG for the sfGFP as described on the figure. Then cells were incubating for 24 h at 18°C after induction until the samples were analyzed with fluorescent microscopy.

Figure 1: Fluorescence microscopy of Encapsulin expression. E. coli (BL21) was co-transformed with EncA and either A) sfGFP or A) sfGFP C-terminally fused to the targeting peptide for the Encapsulin. Arabinose and IPTG induces the expression of EncA and sfGFP respectively. Panels presents GFP fluorescence. sfGFP localizes mostly throughout the cytoplasm in both conditions. Single dots in cells are formed when IPTG is only expressed due to leaky expression. Representive pictures of three replicates.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 47
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 47
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 47
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 107