Part:BBa_K3111103
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 a modification to BBa_K2686002, 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 (Moon et al., 2014). Since these properties seemed attractive from a potential manufacturing perspective, the HexaHistidine tag was kept in the newly designed part.
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 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 in greater purity and solubility.
It is expressed under a T7 promoter and a strong RBS.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 183
Illegal BglII site found at 598 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1013
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 532
Illegal SapI.rc site found at 563
Experimental Results
Scale-up
Day 1
Different batches of BL21 (DE3) competent cells were transformed with pSB1C3 plasmids containing BBa_K3111103 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_K3111103 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_K3111103. The culture was incubated at 37°C until it reached an OD600 of 0.6. Once they reached OD600 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
In-vivo expression and purification of BBa_K2686002
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 </i>in-vivo</i>. Unlike in CFPS production, this previously designed BioBrick was 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 (~35 kDa) in the soluble and purified fractions obtained from the E. coli BL21(DE3) cells. Nevertheless, a band of approximately 35 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. As a result, we decided to clone part BBa_K2686002 with the T7 promoter and RBS that we used in our other constructs.
After expressing with BBa_J64997 and BBa_K2306014 we managed to successfully overexpress T. maritima encapsulins (Figure 2). 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. Additionally, we observed that when expressed without the StrepII tag, the encapsulin is slightly less soluble: in Figure 1, we can note that there is more encapsulin in the soluble fraction than the insoluble fraction, while in Figure 3, expression of insoluble encapsulin seems to be slightly more dominant.
In-vivo Expression and Purification of BBa_K3111103
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 spun 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 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 T. maritima encapsulin monomer with the HexaHistidine linkers soluble when expressed in-vivo. This was evidenced by the SDS PAGE performed in the insoluble, soluble and purified (E2-E4 in Figure 3) 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 E. coli 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 3, E2-4) than heat purification (Figure 2, Lane 8). We hypothesised that higher quantities of encapsulin are made during in-vivo 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 3, soluble and insoluble fractions) than the one without (Figure 2, Lanes 6 and 7).
Dynamic Light Scattering
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 4).
Confirmation of Self-Assembly Using Transmission Electron Microscopy (TEM)
Having characterised the solubility and potential for self-assembly of the monomers encoded by our improved part using SDS PAGE and DLS, we decided to confirm the potential for self-assembly of our expressed protein constructs by visualising them using TEM. The images obtained (Figure 5) confirmed the results that we had obtained after the DLS experiments: encapsulin assembly and formation of aggregates.
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_K2686002 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 BBa_K3111502.
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