Part:BBa_K3111102

T. maritima encapsulin 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_K2686001, which was designed by the EPFL 2018 iGEM team.

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:

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

This BioBrick constitutes a modification to BBa_K2686001, which was designed by the EPFL 2018 iGEM team. Unlike BBa_K2686001, 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 T. maritima encapsulin monomers, a comparative characterisation of the unstudied in vivo expression of the existing part and our modified version of the part concluded that the insertion of the StrepII tag also resulted in greater purity.

This part is an HexaHistidine-lacking version of BBa_K3111103 (4). The HexaHistidine linker (GGGGGGHHHHHHGGGGG) present between residues 43 and 44 of the encapsulin monomers encoded by BBa_K3111103 is absent from those expressed from this part (4). Therefore, the heat stability of the encapsulin multimers originated from the protein monomers encoded by this BioBrick was lower than that of BBa_K3111103-encoded ones (4).

It is expressed under a T7 promoter and a strong RBS.

Experimental Results

Scale-up

Day 1

Different batches of BL21 (DE3) competent cells were transformed with pSB1C3 plasmids containing BBa_K3111102 sequence coding for the T. maritima T=1 encapsulin monomer. Transformed cells were grown in LB agar plates containing chloramphenicol and glucose. Plates were incubated at 37°C overnight.

Day 2

Transformed colonies containing pSB1C3 + BBa_K3111102 were used to prepare overnight starter cultures containing a total of 5 mL LB broth and chloramphenicol (5 μL). Cultures were incubated at 37 °C overnight.

Day 3

A 50 mL scale-up culture was prepared from a single starter culture containing cells carrying pSB1C3 + BBa_K3111102. The culture was incubated at 37°C until it reached an OD of 0.6. Once they reached OD 0.6, the cultures were induced by addition of 400 μΜ IPTG. The cultures were left to grow again overnight at 37 °C.

Day 4

The culture was collected and transferred into a 50 mL Falcon tube. It was spun for 10 minutes at 5000 rpm in order to pellet the cells. Then the supernatant was discarded, and the pellet frozen at -80 °C.

Protein Analysis

Expression and Purification

In order to observe whether our StrepII-tag-containing encapsulin monomers were successfully expressed, we analysed our cell pellet using SDS PAGE. The pellet obtained from the 50 mL cultures was then resuspended in Tris Buffer Saline at an OD600 of 10. Once resuspended, the sample was cell lysed using sonication. Following sonication, the sample were span to separate the soluble and insoluble fragments form the whole cell lysate. 50 μL from each sample were obtained and stained with Laemmli reagent.

We proceeded on with purification of the soluble fragment using column chromatography containing Strep-Tactin 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 T. maritima encapsulin monomers for binding sites with the resin, thus detaching the protein of interest from the column. Finally, we recycled the column ready for future purifications. From each of the samples obtained during the procedure we obtained 50 μL to use for SDS PAGE.

SDS PAGE revealed that BBa_K3111102-encoded encapsulin monomers were absent from the soluble fraction at a post-induction incubation temperature of 37°C (Figure 1A). Nevertheless, in vivo expression of the same BioBrick at a post-induction incubation temperature of 18°C yielded a purifiable monomer yield in the soluble fraction of the cell lysate (Figure 1B).

Figure 1: Figure 1. SDS PAGE of soluble (S) and insoluble (I) fractions and affinity-purified (E1-E6) elutions of the improved HexaHistidine-lacking T. maritima 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 in vivo and was present in the soluble fraction obtained from E. coli BL21(DE3) cultures when these were incubated at a post-induction temperature or 18ºC (B). This was indicated by the presence of a band of approximately 32 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 37ºC (A). This was evidenced by the absence of bands with the size of encapsulin monomers (32 kDa) in the soluble and purified fractions obtained from the E. coli BL21(DE3) cells (red rectangle, 1A). M: PageRuler Protein Ladder, L: Load flowthrough, W: Wash, E1-6: Elution 1-6.

Comparison to purification of BBa_K2686001

We compared the Strep-Tactin column purification method with heat-purification method used by EPFL 2018 iGEM team. First, we attempted to express their original plasmid in vivo. However, as seen in Figure 2, the overexpression of encapsulins was not observed and the majority of the protein was insoluble. As a result, we decided to clone parts BBa_K2686001 and BBa_K2686002 with the T7 promoter and RBS that we used in our other constructs.

Figure 2: Figure 2. SDS PAGE of soluble (S), insoluble (I) and heat-purified (P) fractions of the T. maritima encapsulin monomers expressed in vivo at 37°C (A) and 18°C (B) from existing parts (i.e. BBa_K2686001 and BBa_K2686002). Unlike in CFPS production, these previously designed BioBricks were not expressed as soluble monomers in the soluble fraction of the cell lysate and, consequently, they could not be heat-purified. This was evidenced by the absence of bands with the size of encapsulin monomers (32 kDa) in the soluble and purified fractions obtained from the E. coli BL21(DE3) cells. Nevertheless, a band of approximately 32 kDa was observed in the insoluble fractions of the cell lysates of cultures incubated at a post-induction temperature of 37°C and 18°C. The band size at which encapsulin monomers were present (insoluble fraction) or expected to be (soluble and purified fractions) is indicated by the red rectangles. M: PageRuler Plus Protein Ladder.

After expressing with BBa_J64997 and BBa_K2306014 we managed to successfully overexpress T. maritima encapsulins (Figure 3). Unexpectedly, we found that the heat purification procedure did not produce high purity encapsulins and a lot of native E. coli proteins were still present in the mixture. We concluded that this method would not be effective for clinical applications that we are aiming for with our project.

Figure 3: Figure 3. SDS PAGE of soluble and insoluble fractions and heat-purified samples of BBa_K2686001 and BBa_K2686002, expressed under the same promoter/RBS. (1) Control soluble fraction, (2) Control insoluble fraction, (3) BBa_K2686001 soluble fraction, (4) BBa_K2686001 insoluble fraction, (5) BBa_K2686001 heat purified, (6) BBa_K2686002 soluble fraction, (7) BBa_K2686002 insoluble fraction, (8) BBa_K2686002 heat purified.

Dynamic Light Scattering

Finally, we tested the assembly of our soluble encapsulin monomers performing DLS studies. In agreement with the results from the SDS PAGE gel (which revealed the absence and presence of soluble encapsulin monomers at post-induction incubation temperatures of 37°C and 18°C respectively), it was observed that, cultures incubated at a post-induction temperature of 37ºC did not assemble to form 20-24 nm 60-mer encapsulin shells (1A). On the other hand, self-assembled encapsulin cages were detected by DLS in samples proceeding from cell lysates of cultures incubated at a post-induction temperature of 18°C as we could observe a peak at the expected size (4B, red arrow). We also noticed a high amount of aggregates forming (evidenced by a signal peaking at ≈ 37 nm), which we decided was a consequence of inadequate buffer conditions.

Figure 4: DLS of BBa_K3111102 produced at 37°C (A) and 18°C (B). As expected, given that no soluble encapsulin monomers were produced at 37ºC, no encapsulins could be assembled at that temperature. This was confirmed by the lack of signal at ≈ 20-24 nm (2A), which is the diameter of the 60-mer encapsulin shell into which monomers self-assemble when expressed in T. maritima. Neverhteless, at 18ºC, DLS studies detected the presence of molecular assemblies with sizes ranging 20-100 nm (2B). We speculate that, since encapsulin monomers were expressed in the soluble fraction of our cell lysates (see Figure 1B), a proportion of it would self-assemble into 60-mer cages of approximately 20-24 nm in diameter and would, subsequently, originate some of the broad signal observed in DLS assessment.

Conclusion

Integration of the results obtained from the SDS PAGE studies confirmed that the modifications that we had introduced into the previously designed BioBrick BBa_K2686001 had improved it by adding a chromatographic method that could be employed to purify encapsulins and increasing the purity of final product by utilizing this method.

References

[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.

[2] Giessen TW, Silver PA. Widespread distribution of encapsulin nanocompartments reveals functional diversity. Nat Microbiol. 6 de marzo de 2017;2:17029.

[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.

[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.