Part:BBa_K4229069
Encapsulin with RBS
This biobrick showes the encapsulin(BBa_K4229020) with its RBS.
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 visualisation 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 in the figure. Then cells were incubated for 24 h at 18°C after induction until the samples were analysed with fluorescent microscopy.
From the fluorescent microscopy, see Figure 1, the expression of Encapsulins in the 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.
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.
[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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
None |