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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4229069 short</partinfo>
-Here we have the encapsuline(BBa_K4229020) with its RBS(BBa_K4229004). Encapsulins  are nanocompartments, which similar to microcompartments, are self-assembled protein compartments, natively found in some bacteria and archaea [1]. They can be distinguished from microcompartments through the size of the compartments (20-42 nm) [1]. The encapsulins from <i>Thermotoga maritima</i> and <i>Mycobacterium tuberculosis</i> have outer diameters of 20–24 nm and are composed of 60 identical encapsulin protein subunits. The largest encapsulin compartment discovered to date is represented by that of Quasibacillus thermotolerans with a 42 nm outer diameter and 240 identical subunits [2]. Structural experiments showed that encapsulins are icosahedral shell-like protein compartments resembling viral capsids (Hong Kong 97‐like fold) [2][3]. The pore size of ~5 Å allows channelling small molecular substrates through the shell. Multiple encapsulins encapsulate cargo protein based on a short C-terminal peptide sequence, called the targeting peptide (TP) [1]. TPs often include a specific anchoring sequence, such as the Gly–Ser–Leu singlet or doublet motif and binding is mediated by hydrophobic and ionic interactions [4][6]. Encapsulins have attracted the attention of the synthetic biology community for the possibility of engineering small protein nanocages e.g. for drug delivery [5]. The encapsulins are highly suitable for such purposes given their high stability at high temperatures and various pH levels [8]. For our project, we decided to use an encapsulin derived from <i>M. xanthus</i> which is composed of the protein EncA, forming the shell [3][7]. In <i>M. xanthus</i>, the encapsulin is known to encapsulate three different cargo proteins, which play a role in iron storage [7]. This specific encapsulin was deemed a great fit for our team, as it has previously been engineered to encapsulate non-native enzymes in yeast [9]. For exmperimental data look into the Biobrick BBa_K4229070.
+This biobrick consists the encapsulin (BBa_K4229020) with its RBS.
-[[File:Encapsulines.png|200px|thumb|left|Encapsulin of the organism M. xanthus, which is composed of the protein EncA. Encapsulin compartments can provide stabilization and co-localization of cargo proteins and can also be engineered to encapsulate non-native enzymes, as previously shown in yeast (structure from PDB: 4PT2). Figure created with PyMOL.]]
+Usage:
+Nanocompartments are found in some bacteria and archea. The encapsulin natively expressed in <i>M. Xanthus</i> is composed of the protein EncA [1][2]. In <i>M. Xanthus</i>, 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 <i>in vivo</i>.
-[1] T. W. Giessen, “Encapsulins: Microbial nanocompartments with applications in biomedicine, nanobiotechnology and materials science,” Curr. Opin. Chem. Biol., vol. 34, pp. 1–10, 2016, doi: 10.1016/j.cbpa.2016.05.013.
+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.
-[2]J. A. Jones and T. W. Giessen, “Advances in encapsulin nanocompartment biology and engineering,” Biotechnol. Bioeng., vol. 118, no. 1, pp. 491–505, 2021, doi: 10.1002/bit.27564.
+[[File:SfGF1.jpg|800px|thumb|left|Figure 1: Fluorescence microscopy of Encapsulin expression. <i>E. coli</i> (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.]]
-[3]	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.
-[4]	M. Sutter et al., “Structural basis of enzyme encapsulation into a bacterial nanocompartment,” Nat. Struct. Mol. Biol., vol. 15, no. 9, pp. 939–947, 2008, doi: 10.1038/nsmb.1473.
-[5]	A. Van de Steen et al., “Bioengineering bacterial encapsulin nanocompartments as targeted drug delivery system,” Synth. Syst. Biotechnol., vol. 6, no. 3, pp. 231–241, 2021, doi: 10.1016/j.synbio.2021.09.001.
-[6]	W. J. Altenburg, N. Rollins, P. A. Silver, and T. W. Giessen, “Exploring targeting peptide-shell interactions in encapsulin nanocompartments,” Sci. Rep., vol. 11, no. 1, pp. 1–9, 2021, doi: 10.1038/s41598-021-84329-z.
-[7]	C. A. McHugh et al., “A virus capsid‐like nanocompartment that stores iron and protects bacteria from oxidative stress,” EMBO J., vol. 33, no. 17, pp. 1896–1911, 2014, doi: 10.15252/embj.201488566.
-[8]	I. Boyton, S. C. Goodchild, D. Diaz, A. Elbourne, L. Collins-Praino, and A. Care, “Exploring the Self-Assembly of Encapsulin Protein Nanocages from Different Structural Classes,” bioRxiv, 2021, doi: 10.1101/2021.06.06.447285.
-[9]	Y. H. Lau, T. W. Giessen, W. J. Altenburg, and P. A. Silver, “Prokaryotic nanocompartments form synthetic organelles in a eukaryote,” Nat. Commun., vol. 9, no. 1, 2018, doi: 10.1038/s41467-018-03768-x.
+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.
+[[File:SfGFPEnc2.jpg|800px|thumb|left|Figure 2: Fluorescence microscopy of Encapsulin expression. <i>E. coli</i> (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.
+[[File:SfGFPEnc3.jpg|800px|thumb|left|Figure 3: Fluorescence microscopy of Encapsulin expression. <i>E. coli</i> (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.