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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 183
  Illegal BglII site found at 598
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1013
  
• 1000
  INCOMPATIBLE 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 E. coli 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

Replication of Cell-Free Protein Synthesis (CFPS) of BBa_K2686002

First, we decided to replicate the cell-free synthesis and heat purification of T. maritima encapsulins that we received from plasmids sent by Team EPFL. We used the <a href="http://2018.igem.org/Team:EPFL/Protocols">protocols</a> provided by them (albeit with some minor changes for the CFPS portion) and expressed parts BBa_K2686001 and BBa_K2686002. From Figure 1, we can note that encapsulins we expressed (bands indicated by red arrows, encapsulin with the 6-His insert was expected to be slightly bigger), however, no overexpression was observed. Unexpectedly, the heat purification did not yield pure T. maritima encapsulin. Regardless, we proceeded to work with the plasmid containing BBa_K2686002 further on as expression was verified.

Figure 1: SDS PAGE of CFPS and heat purification of T. maritima encapsulins (indicated by the red arrows). M: molecular marker, 1: CFPS of T. maritima encapsulin; 2: Heat purification of T. maritima encapsulin; 3: CFPS of T. maritima encapsulin with 6-His insert; 4: Heat purification of T. maritima encapsulin with 6-His insert.

In-vivo expression and purification of BBa_K2686002

We compared the Strep-Tactin^TM 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.

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 the existing part BBa_K2686002. 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^TM 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.

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^TM 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 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 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 4, E2-4) than heat purification (Figure 4, 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 4, soluble and insoluble fractions) than the one without (Figure 3, Lanes 6 and 7).

Figure 4: Figure 4. 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<i> and was present in the soluble fraction obtained from <i>E. coli 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 E. coli BL21(DE3) cells (red rectangle, 1A). M: PageRuler^TM Protein Ladder, L: Load flowthrough, W: Wash, E1-6: Elution 1-6.

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 5).

Figure 5: DLS of the improved HexaHistidine-containing T. maritima encapsulin monomers produced 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).

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 6) confirmed the results that we had obtained after the DLS experiments: encapsulin assembly and formation of aggregates.

Figure 6: TEM image of the assembled encapsulins that were purified from the soluble fraction of <i>E. coli BL21(DE3) bacterial lysates applying Strep-tag affinity chromatography. As expected, the estimated diameter of the encapsulin shells that were visualised in the TEM images was approximately 20-24 nm.

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.