Difference between revisions of "Part:BBa K2686001"

Latest revision as of 19:37, 13 October 2019

Encapsulin protein

This is a BioBrick containing the sequence for Thermotoga maritima encapsulin from David Savage (Addgene #86405), a bacterial protein nanocompartment which self assembles to form a 60-mer.

Usage and Biology

Encapsulins are versatile proteins found in a variety of different bacteria (Giessen and Silver, 2017). In the case of this specific part derived from Thermotoga maritima, it can be used among other things to deliver cargo, both on the outer surface of the nanoparticle by fusing a peptide in between the 139/140 Amino Acids or the protein's C terminus. A cargo protein can also be loaded by fusing it with a tag binding to Encapsulin's interior surface (Cassidy-Amstutz et al., 2016).

Sequence and Features

Characterization

A variety of different characterization techniques were used to assess the properties of the encapsulin protein cage.

Expression

A cell free expression system was used to synthesize the encapsulin proteins in vitro. The TX-TL cell free system is a robust way to express proteins (Sun et al., 2013), and we used the protocol developed by the 2017 EPFL iGEM team Aptasense.

SDS PAGE of encapsulins expressed in cell free TX-TL system with Lysine-BODIPY fluorescent tRNA's (Imager settings used were for measuring fluorescein λ_ex=494nm, λ_em=512nm ). The two different sets of lanes correspond to different heat denaturation temperatures (70C and 100C for 15 minutes). Stars (✦) show lanes where 60-mers are present. From left to right: (L) Positive control with DNA coding for Luciferase (37kDa), (H) HexaHistidine Encapsulin (BBa_K2686002) showing bands for the encapsulin multimer high on the gel lanes (✦) as well as the monomer around 31kDa, (R) Encapsulin (BBa_K2686001) without HexaHistidine linker also has a band for the 60-mer (✦), (N) Negative control (cell-free TX-TL expression without DNA and 100C denaturation), (Ladder) LC5928 BenchMark™ Fluorescent Protein Standard, (L) Positive control with DNA coding for Luciferase (37kDa), (H) HexaHis Encapsulin (BBa_K2686002) showing bands for the encapsulin multimer high on the gel lanes (✦) as well as the monomer around 31kDa, (R) Encapsulin (BBa_K2686001) without HexaHistidine linker also showing the multimer (✦), (N) Negative control (cell-free TX-TL expression without DNA and 70C denaturation)

Purification

After having tested a variety of purification procedures, heat purification at 70C for 20 minutes followed by cooling on ice for 15 minutes and a subsequent centrifugation at 12000g for 10 minutes was found to be the most efficient way of isolating the encapsulin (encapsulin ends up in supernatant).

Assembly

The self assembly of the encapsulin 60-mer was first examined using SDS PAGE, where a band around 30.71kDa band is expected to form. Additionally due to Encapsulin's exceptional heat stability the 1.98MDa complex also appears on the gel after SDS denaturation.

Native PAGE gel of sfGFP, HexaHistidine-OT1 Encapsulin and HexaHistidine-OT1 Encapsulin sfGFP-tag. On the left is the fluorescent image of the gel and o the right is the gel after Coomassie staining. Before (B) heat purification procedure outlined in Purification section, Supernatant (S) after the heat purification procedure. Stars ★ show the bands for monomers of HexaHistidine-OT1 Encapsulin (BBa_K2686000,BBa_K2686006) with sfGFP-tag bound to them, increasing the molecular weight of the complex when compared to sfGFP alone. Triangles ▲ signify a 60-mer band to the right. From left to right: (1-2) Negative control (cell-free TX-TL expression without DNA), (2-4) HexaHistidine-OT1 Encapsulin (BBa_K2686000), showing no fluorescence on the left gel, has a band for 60-mer on stained gel (▲). (5-6) sfGFP protein, the fluorescent bands can be easily seen as large smears on the left. (7-8) HexaHistidine-OT1 Encapsulin and sfGFP-tag (BBa_K2686006) shown by a ★. The difference in height between the bands of sfGFP compared to the ★ is striking and suggests that the sfGFP-tag binds to the HexaHistidine-OT1 Encapsulin monomers. In addition there seems to be a small amount of 60-mer indicated by ▲. (9-10) Encapsulin (BBa_K2686001) does assemble to form the 60-mer ▲ as seen on the stained gel. (11) HexaHistidine-OT1 Encapsulin and sfGFP-tag (BBa_K2686006), on the left the presumed ★ protein dimer is seen to be higher than the sfGFP in lane 12, no particular bands can be identified on the right. (12) sfGFP, fluorescent band is seen to be lower than for lane 11.

In vivo Characterisation by Team UCL 2019

Scale-up Procedure

Day 1

Different batches of BL21(DE3) competent cells were transformed with pSB1C3 plasmids containing BBa_K2686001 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_K2686001 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_K2686001. 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.

Expression Analysis

1. Expression In order to observe whether BBa_K2686001-encoded 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. According to our expression tests, this part was not suitable for successful production and purification of T. maritima encapsulin monomers in E. coli BL21(DE3). In vivo encapsulin production was hindered by the aggregation of the protein monomers at different production temperatures (i.e. 37ºC and 18ºC). This was evidenced by the SDS-PAGE gels which we run to test the presence of T. maritima encapsulin monomers in the insoluble, soluble and heat-purified fractions obtained from our cell lysates. As shown in Figure 1, T. maritima encapsulin monomers (~ 32 kDa), which were produced in vivo by E. coli BL21(DE3), were concentrated in the insoluble fraction of BBa_K2686001- expressing bacteria even when the temperature of post-induction incubation was decreased from 37ºC (Figure 1A) to 18ºC (Figures 1B) to favour protein expression.

Figure 1: SDS-PAGE of soluble (S), insoluble (I) and purified (P) fractions of the T. maritima encapsulin monomers expressed in vivo at 37ºC (A) and 18ºC (B) from BBa_K2686001. Unlike in CFPS production, this BioBrick was not expressed as soluble monomers in the soluble fraction of the cell lysate and, consequently, these monomers 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 37ºC and 18ºC. 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= molecular marker, L= flow-through fraction obtained after loading the sample into the column and W= fraction generated after adding binding buffer to the column immediately after the sample loading and L elution collection.

Self-Assembly

Due to the lacking or low-level solubility of T. maritima encapsulin monomers produced by in vivo expression of this part, these could not assemble to form the loadable 60-mer protein shells in in vivo systems. This was evidenced by dynamic light scattering (DLS). Under normal (i.e. soluble) conditions, T. maritima T=1 encapsulin monomers self-assemble into an encapsulin cage with a diameter of approximately 20-24 nm. Nevertheless, after performing DLS in the heat-purified soluble fractions obtained from BBa_K2686001-expressing bacteria, we observed that, at both temperatures of post-induction incubation (i.e. 37ºC and 18ºC), no signal was obtained for molecules ranging that size in the samples used. Instead, as displayed in Figure 2, DLS studies only detected the presence of monomers (diameter ~ 1 nm) and aggregates (diameter > 100 nm) of BBa_K2686001-encoded T. maritima encapsulin monomers at both production temperatures. Thus, DLS confirmed that, as it was observed from the SDS-PAGE gels, BBa_K2686001-encoded encapsulin monomers were mostly insoluble in in vivo protein expression systems and unable to self- assemble into T. maritima encapsulins.

Figure 2: DLS of the heat-purified fractions obtained from cell lysates of E. coli BL21(DE3) expressing T. maritima encapsulin monomers. The bacterial cultures were incubated at a post- induction temperature of 37ºC (A) and 18ºC (B). The peaks revealed the presence of monomers (diameter ~ 1 nm) and aggregates (diameter > 100 nm) of the protein construct. The absence of a signal at 20-24 nm indicated that there were no assembled 60-mers encapsulin cages in the heat-purified fractions.

Conclusion

We hypothesised that there may be some problems in T. maritima encapsulin in vivo isolation when applying the heat purification method for which BBa_K2686001 had originally been designed. Firstly, a few other bacterial proteins may be stable at the temperature at which the heat-purification was performed and, subsequently, co-purify with the encapsulins that were being targeted. In fact, we observed that this was the case in cell-based protein expression systems, as, in the SDS-PAGE gel, we detected bands indicating the presence of proteins with molecular weights different to 32 kDa (Figure 1). This revealed that not only the encapsulin monomers had been heat purified and, therefore, there were additional proteins present in the purified analysed sample. This unspecific protein co-purification would not only reduce the purity of our target protein in the purified soluble sample but could even contribute to lowering the solubility of the previously existing parts coding for encapsulin monomers (Figures 1 and 2).

To overcome the challenges that arose with the in vivo solubility and self-assembly capacity of the monomers encoded by this part, the UCL 2019 iGEM team re-evaluated the purification by expressing BBa_K2686002 with T7 promoter (BBa_J64997) and a strong RBS (BBa_K2306014). After showing that said features allowed protein expression in vivo, Team UCL 2019 designed BBa_K3111102. which encodes T. maritima encapsulin monomers with an inserted StrepII tag that allowed for in vivo expression and purification using affinity chromatography.

References

Cassidy-Amstutz, C., Oltrogge, L., Going, C., Lee, A., Teng, P., Quintanilla, D., East-Seletsky, A., Williams, E. and Savage, D. (2016). Identification of a Minimal Peptide Tag for in Vivo and in Vitro Loading of Encapsulin. Biochemistry, 55(24), pp.3461-3468.

Giessen, T. and Silver, P. (2017). Widespread distribution of encapsulin nanocompartments reveals functional diversity. Nature Microbiology, 2, p.17029.