Part:BBa_K4229061

sfGFP with targetpeptide for encapsulins regulated by T7 promoter

This Biobrick shows an sfGFP version onto which a target peptide was added. This makes it possible to recruit this sfGFP into encapsulines (BBa_K4229061) and use the fluorescence as a method to see the recruitment.

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.

From the fluorescent microscopy, see Figure 1, the expression of Encapsulins in presence of sfGFP for fluorescent “foci” believed to be the encapsulated sfGFP in Encapsulins. At expression levels beyond leaky expression, the occurrence of fluorescent “foci” decreased, suggesting that these are not due to inclusion bodies. At higher expression levels of Encapsulins, see Figure 2, the fluorescent foci are more easily detectable. However, at high concentrations of sfGFP the amount of fluorescent “foci” decreases.

Figure 2:Fluorescence microscopy of Encapsulin expression. E. coli (BL21) was co-transformed with EncA and either sfGFP or sfGFP C-terminally fused to the targeting peptide for the Encapsulin. Arabinose and IPTG induces the expression of EncA and sfGFP respectively. sfGFP localizes mostly throughout the cytoplasm while sfGFP_TP forms fluorescent “foci”.

Control experiment shows the co-expression of pBAD33, the backbone of the EncA, and either sfGFP or sfGFP_TP. Figure 3 shows that the sfGFP fluorescence is mainly located throughout the cytoplasma. Occasionally single fluorescent “foci” are formed, but not to the same degree as when Encapsulins are expressed.

Figure 3: Fluorescence microscopy of Encapsulin expression. E. coli (BL21) was co-transformed with pBAD33 and either A) sfGFP or A) sfGFP C-terminally fused to the targeting peptide for the Encapsulin. Arabinose and IPTG induces the expression of pBAD33 and sfGFP respectively. Panels presents GFP fluorescence. sfGFP localizes mostly throughout the cytoplasm in both conditions. Single dots are formed occasionally.

[1] J. Fontana et al., “Phage capsid-like structure of Myxococcus xanthus encapsulin, a protein shell that stores iron,” Microsc. Microanal., vol. 20, no. 3, pp. 1244–1245, 2014, doi: 10.1017/S1431927614007958.

[2] F. Johnsson, J. Kjärstad, and J. Rootzén, “The threat to climate change mitigation posed by the abundance of fossil fuels,” Clim. Policy, vol. 19, no. 2, pp. 258–274, 2019, doi: 10.1080/14693062.2018.1483885.

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