Difference between revisions of "Part:BBa K4229069"

Latest revision as of 22:53, 13 October 2022

Encapsulin with RBS

This biobrick consists 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]. Cargos can be targeted to the encapsulin through a native targeting peptide (TP) 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. Bacterial 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 shown in the Figure 1. Then cells were incubated for 24 h at 18°C after induction before with fluorescent microscopy.

Figure 1: Fluorescence microscopy of Encapsulin expression. E. coli (BL21) was co-transformed with EncA and either A) sfGFP or B) sfGFP C-terminally fused to the targeting peptide (TP) for the Encapsulin. Arabinose and IPTG induce 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. Representative pictures of three replicates.

From the fluorescent microscopy (Figure 1), expression of Encapsulins in the presence of sfGFP form fluorescent foci believed to be the encapsulated sfGFP. 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 (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 with a C-terminal targeting peptide (TP). 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, co-expression of pBAD33, the backbone of the EncA, and either sfGFP or sfGFP_TP shows that the sfGFP fluorescence is mainly located throughout the cytoplasma (Figure 3). Occasionally, single fluorescent “foci” are formed, but not to the same degree as when co-expressed with Encapsulins.

Figure 3: Fluorescence microscopy of Encapsulin expression. E. coli (BL21) was co-transformed with pBAD33 and either A) sfGFP or A) sfGFP with a C-terminal targeting peptide (TP). 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.

References:

[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