Difference between revisions of "Part:BBa K3111103"
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<partinfo>BBa_K3111103 short</partinfo> | <partinfo>BBa_K3111103 short</partinfo> | ||
| − | This part encodes for the <i>Thermotoga maritima</i> T=1 encapsulin monomer, which, once expressed, self-assembles with additional monomers into a 60-mer encapsulin shell with a diameter of approximately 20-24 nm. | + | This part encodes for the <i>Thermotoga maritima</i> T=1 encapsulin monomer, which, once expressed, self-assembles with additional monomers into a 60-mer encapsulin shell with a diameter of approximately 20-24 nm. This BioBrick constitutes an improvement to <partinfo>BBa_K2686002</partinfo>, which was designed by the EPFL 2018 iGEM team. |
| − | + | <!-- Add more about the biology of this part here | |
| + | ===Usage and Biology=== | ||
| − | + | Research into bacterial metabolic compartmentalisation has led to discovery of a new class of bacterial proteinaceous structures named Bacterial Microcompartments (BMCs), which act in an analogous fashion to eukaryotic organelles(1). | |
| − | + | In the mid-1990s, a new-class of structurally smaller and sequence dissimilar compartments called encapsulins within Brevibacterium Linens was discovered followed by the Pyrococcus furiosus and Thermotoga maritima encapsulins which revealed their capsid-like morphology loaded with cargo-proteins. To date more than 900 encapsulin systems with a diversity of putative cargo proteins have been identified spanning a remarkable breadth of microbial diversity and habitats(2). | |
| + | High resolution crystallography has revealed that encapsulin nano-compartments are assembled entirely from a single protein protomer, making them distinct from the conventional eukaryotic organelles. The shell proteins are homologs of HK97 viral phages and assemble into defined icosahedral architectures of varying size depending on the species(3). They are classified into 2 classes determined from their triangulation number (T). T=1 encapsulins form smaller structures (20-24 nm in diameter) composed of 60 protomers while T=3 encapsulins are bigger (30-32 nm in diameter) and are composed of 180 monomers. Furthermore, they have multiple pores of 5–6 Å of varying chemical nature that control the exchange of small molecules between the cytosol and the lumen facilitating their metabolic function. | ||
| − | + | Research literature indicates 2 major roles of encapsulins: | |
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| − | + | Bacterial protection against environmental stresses - especially oxidative and nitrosative stresses - by forming complexes with ferritin-like enzymatic proteins and hemerythrins(3). | |
| − | + | Participation in the global nitrogen cycle as they were identified within anammox bacteria. | |
| − | + | However, their presence in many less explored phyla like the Planctomycetes and Tectomicrobia might reveal other novel functions as this field moves forward. | |
| + | ===Functionalisation Parameters=== | ||
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| + | This BioBrick constitutes a modification to <partinfo>BBa_K2686002</partinfo>, which was designed by the EPFL 2018 iGEM team. Like its previously existing counterpart, our BioBrick codes for a HexaHistidine linker (GGGGGGHHHHHHGGGGG) between residues 43 and 44. This sequence had been shown to increase the heat stability of the encapsulin multimer (4). Since these properties seemed attractive from a potential manufacturing perspective, the HexaHistidine tag was kept in the newly designed part. | ||
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| + | However, unlike BBa_K2686002, our construct encodes an inserted StrepII tag to purify assembled encapsulins without heat treatment. Although this modification was originally expected to only expand the variety of chromatographic methods that could be applied to purify <i>T. maritima</i> encapsulin monomers, a comparative characterisation of the unstudied <i>in-vivo</i> expression of the existing part and our modified version of the part concluded that the insertion of the StrepII tag also resulted in in greater purity and solubility. | ||
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| + | It is expressed under a T7 promoter and a strong RBS. | ||
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==Experimental Results== | ==Experimental Results== | ||
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We proceeded on with purification of the soluble fragment using column chromatography containing Strep-Tactin<sup>TM</sup> resin. The process involved packing the column, equilibrating the resin and loading the soluble sample. Then a washing step was performed to remove any potential non bound nonspecific proteins. Then we eluted using competitive elution by loading BXT which competed with the <i>T. maritima</i> encapsulin monomers for binding sites with the resin, thus detaching the protein of interest from the column. Finally, we recycled the resin for future purifications. From each of the samples obtained during the procedure we obtained 50 μL to use for SDS PAGE. | We proceeded on with purification of the soluble fragment using column chromatography containing Strep-Tactin<sup>TM</sup> resin. The process involved packing the column, equilibrating the resin and loading the soluble sample. Then a washing step was performed to remove any potential non bound nonspecific proteins. Then we eluted using competitive elution by loading BXT which competed with the <i>T. maritima</i> encapsulin monomers for binding sites with the resin, thus detaching the protein of interest from the column. Finally, we recycled the resin for future purifications. From each of the samples obtained during the procedure we obtained 50 μL to use for SDS PAGE. | ||
| − | The construct coding for the <i>T. maritima</i> encapsulin monomer with the HexaHistidine linkers soluble when expressed <i>in-vivo</i>. This was evidenced by the SDS PAGE performed in the insoluble, soluble and purified (E2-E4 in Figure 4) fractions of cell lysates obtained from cultures which had been incubated at a post-induction temperature of 37°C. Therefore, although there was no need to further enhance the solubility of the monomers in this instance, the expression of the improved constructs could potentially be enhanced by incubating the <i>E. coli</i> BL21 (DE3) cultures at a post-induction temperature which favours protein expression (i.e. 18°C). | + | The construct coding for the <i>T. maritima</i> encapsulin monomer with the HexaHistidine linkers soluble when expressed <i>in-vivo</i> (4). This was evidenced by the SDS PAGE performed in the insoluble, soluble and purified (E2-E4 in Figure 4) fractions of cell lysates obtained from cultures which had been incubated at a post-induction temperature of 37°C. Therefore, although there was no need to further enhance the solubility of the monomers in this instance, the expression of the improved constructs could potentially be enhanced by incubating the <i>E. coli</i> BL21 (DE3) cultures at a post-induction temperature which favours protein expression (i.e. 18°C). |
Comparing the constructs with and without the StrepII-tag we can see two differences. First, the purity of proteins was evidently higher using column purification (Figure 4, E2-4) than heat purification (Figure 4, Lane 8). We hypothesised that higher quantities of encapsulin are made during <i>in-vivo</i> expression, which in turn makes heat transfer more difficult, lowering the efficiency of heat purification. Second, the construct with a StrepII-tag appeared to be slightly more soluble (Figure 4, soluble and insoluble fractions) than the one without (Figure 3, Lanes 6 and 7). | Comparing the constructs with and without the StrepII-tag we can see two differences. First, the purity of proteins was evidently higher using column purification (Figure 4, E2-4) than heat purification (Figure 4, Lane 8). We hypothesised that higher quantities of encapsulin are made during <i>in-vivo</i> expression, which in turn makes heat transfer more difficult, lowering the efficiency of heat purification. Second, the construct with a StrepII-tag appeared to be slightly more soluble (Figure 4, soluble and insoluble fractions) than the one without (Figure 3, Lanes 6 and 7). | ||
| − | [[Image:TmEncH1.png|700px|thumb|center|'''Figure 4:''' Figure 4. SDS PAGE of soluble (S) and insoluble (I) fractions and affinity-purified (E1-E6) elutions of the improved HexaHistidine-lacking <i>T. maritima</i> encapsulin monomers. The improved part encoded an encapsulin monomer with an inserted StrepII tag which allowed the successful purification of the protein from the soluble fraction of the cell lysate after applying Strep-tag chromatography. Unlike its previously designed counterpart, our improved BioBrick (BBa_K3111102) could be expressed <i>in-vivo<i> and was present in the soluble fraction obtained from <i>E. coli</i> BL21(DE3) cultures when these were incubated at a post-induction temperature or 18°C (B). This was indicated by the presence of a band of approximately 35 kDa in the soluble, insoluble and the first elutions of the purified fraction samples that were run in the SDS PAGE gel (red rectangle, 1B). However, our protein construct was still insoluble when it was expressed at 37°C (A). This was evidenced by the absence of bands with the size of encapsulin monomers (35 kDa) in the soluble and purified fractions obtained from the <i>E. coli</i> BL21(DE3) cells (red rectangle, 1A). M: PageRuler<sup>TM</sup> Protein Ladder, L: Load flowthrough, W: Wash, E1-6: Elution 1-6.]] | + | [[Image:TmEncH1.png|700px|thumb|center|'''Figure 4:''' Figure 4. SDS PAGE of soluble (S) and insoluble (I) fractions and affinity-purified (E1-E6) elutions of the improved HexaHistidine-lacking <i>T. maritima</i> encapsulin monomers (4). The improved part encoded an encapsulin monomer with an inserted StrepII tag which allowed the successful purification of the protein from the soluble fraction of the cell lysate after applying Strep-tag chromatography. Unlike its previously designed counterpart, our improved BioBrick (BBa_K3111102) could be expressed <i>in-vivo<i> and was present in the soluble fraction obtained from <i>E. coli</i> BL21(DE3) cultures when these were incubated at a post-induction temperature or 18°C (B). This was indicated by the presence of a band of approximately 35 kDa in the soluble, insoluble and the first elutions of the purified fraction samples that were run in the SDS PAGE gel (red rectangle, 1B). However, our protein construct was still insoluble when it was expressed at 37°C (A). This was evidenced by the absence of bands with the size of encapsulin monomers (35 kDa) in the soluble and purified fractions obtained from the <i>E. coli</i> BL21(DE3) cells (red rectangle, 1A). M: PageRuler<sup>TM</sup> Protein Ladder, L: Load flowthrough, W: Wash, E1-6: Elution 1-6.]] |
===Dynamic Light Scattering=== | ===Dynamic Light Scattering=== | ||
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Finally, we tested the assembly of our soluble encapsulin monomers performing DLS studies. Although DLS revealed the presence of monomers (diameter ~ 1 nm) and aggregates (diameter > 100 nm), some encapsulins were assembled. This was detected by the presence of peaks at diameter ≈ 20 nm (see Figure 5). | Finally, we tested the assembly of our soluble encapsulin monomers performing DLS studies. Although DLS revealed the presence of monomers (diameter ~ 1 nm) and aggregates (diameter > 100 nm), some encapsulins were assembled. This was detected by the presence of peaks at diameter ≈ 20 nm (see Figure 5). | ||
| − | [[Image:TmEncH2.png|700px|thumb|center|'''Figure 5:''' DLS of the improved HexaHistidine-containing <i>T. maritima</i> encapsulin monomers produced <i>in-vivo<i>. In agreement with the results from the SDS PAGE gel (which revealed the presence of soluble encapsulin monomers at post-induction incubation temperatures of 37ºC), it was observed that, 60-mer encapsulin monomers self-assemble into shells with a diameter of, approximately 20-24 nm (red arrow, red rectangle).]] | + | [[Image:TmEncH2.png|700px|thumb|center|'''Figure 5:''' DLS of the improved HexaHistidine-containing <i>T. maritima</i> encapsulin monomers produced <i>in-vivo<i> (4). In agreement with the results from the SDS PAGE gel (which revealed the presence of soluble encapsulin monomers at post-induction incubation temperatures of 37ºC), it was observed that, 60-mer encapsulin monomers self-assemble into shells with a diameter of, approximately 20-24 nm (red arrow, red rectangle).]] |
===Confirmation of Self-Assembly Using Transmission Electron Microscopy (TEM)=== | ===Confirmation of Self-Assembly Using Transmission Electron Microscopy (TEM)=== | ||
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Integration of the results obtained from the SDS PAGE studies confirmed that the modifications that we had introduced into the previously designed BioBrick <partinfo>BBa_K2686002</partinfo> had improved it by (i) adding a chromatographic method that could be employed to purify encapsulins, (ii) increasing the purity of final product by utilizing this method and (iii) slightly increasing encapsulin solubility. The StrepII tag purification was later used to purify other encapsulin fusion products such as <partinfo>BBa_K3111502</partinfo>. | Integration of the results obtained from the SDS PAGE studies confirmed that the modifications that we had introduced into the previously designed BioBrick <partinfo>BBa_K2686002</partinfo> had improved it by (i) adding a chromatographic method that could be employed to purify encapsulins, (ii) increasing the purity of final product by utilizing this method and (iii) slightly increasing encapsulin solubility. The StrepII tag purification was later used to purify other encapsulin fusion products such as <partinfo>BBa_K3111502</partinfo>. | ||
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| + | ===References=== | ||
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| + | [1] Nichols RJ, Cassidy-Amstutz C, Chaijarasphong T, Savage DF. Encapsulins: molecular biology of the shell. Crit Rev Biochem Mol Biol. 3 de septiembre de 2017;52(5):583-94. | ||
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| + | [2] Giessen TW, Silver PA. Widespread distribution of encapsulin nanocompartments reveals functional diversity. Nat Microbiol. 6 de marzo de 2017;2:17029. | ||
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| + | [3] Giessen TW. Encapsulins: microbial nanocompartments with applications in biomedicine, nanobiotechnology and materials science. Synth Biol Synth Biomol. 1 de octubre de 2016;34:1-10. | ||
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| + | [4] Moon H, Lee J, Min J, Kang S. Developing Genetically Engineered Encapsulin Protein Cage Nanoparticles as a Targeted Delivery Nanoplatform. Biomacromolecules. 2014 Oct 13;15(10):3794–801. | ||
Revision as of 01:55, 19 October 2019
T. maritima encapsulin (6-His) with a StrepII-tag expressed under T7 promoter
This part encodes for the Thermotoga maritima T=1 encapsulin monomer, which, once expressed, self-assembles with additional monomers into a 60-mer encapsulin shell with a diameter of approximately 20-24 nm. This BioBrick constitutes an improvement to BBa_K2686002, which was designed by the EPFL 2018 iGEM team.