HRT2trunc-NT-ASIP

Imperial-College 2024

This part is designed by Team Imperial_College in iGEM 2024. It is a recombinant protein that enables the formation of artificial organelles in E. coli through a phase-separation mechanism inspired by the formation of Spider-silk proteins ^[1].

This basic part encodes the fusion protein of the recombinant rubber cis-polyprenyltransferase HRT2trunc (BBa_K5416000), fused with the N-Terminus domain of spider-silk spidroin protein from Euprosthenops australis (BBa_K3264000) and a hydrophobic peptide (BBa_K5416071) adopted from surfactant protein C33Leu ^[1][2].

Background

Spider silk protein is an excellent example of phase-separated capsulein its native state inside the cell. Further studies into the structure of the protein reveals two major domains: N-terminus hydrophilic domain (NT)that is zwitterionically charged on the two ends, and a hydrophobic domain that is glycine-rich and contains mostly of non-polar amino acids. When translated into the cytosolic environment, the hydrophobic region aggregates inward where the zwitterionic property of the NT allowed efficient insertion of newly made spider protein to be inserted into the aggregated particle.

Fig 1: The proposed formation of spider silk micelle-like particle through interactions between intrinsically disordered peptides (b). The micelle formation has been reported through extent literature (c)(d). Proposed structure of fusion protein with intrinsically disordered region replaced with other hydrophobic peptide would enable the formation of micelle-like structure (e).^[1]

In a recent biomimetic design, the micelle is recreated with the hydrophobic domain replaced with a short leucine/isoleucine rich helix (C33Leu) rod from a hydrophobic peptide hormone. The combination of NT with C33Leu has been shown in previous work to be capable of forming a membrane free micelle.^[1]

Inspired by this engineered spider silk protein micelle, we questioned if the hydrophobic nature of this membrane free micelle could be used as an ideal environment for growing long chain natural rubber. Therefore, we have designed fusion protein which would be able to form the similar structure yet allowing the HRT2trunc protein to be attaching at the surface of micelle and positioning the growth of natural rubber chain inward. In this way, an artificial organelle could be made.

Fig 2: the proposed micelle formation mechanism of this part. Created with Biorender.

Design

Rational Design of the Part's Sequence

Here we report our designed fusion protein HRT2trunc-NT-asip to be consisting of three major domains. The first domain HRT2trunc (BBa_K1088003) is the truncated rubber synthase enzyme derived from HRT2 from Pára rubber tree H. brasiliensis, which has also been characterized by us. The second domain NT corresponds to the N-terminus domain of the spidroin protein from Euprosthenops australis (BBa_K3264000), provides a hydrophilic terminus for forming the outer surface of the micelle. The final domain amphipathic spider inspired protein (ASIP) that is an artificial surfactant protein C33Leu, exhibiting excellent hydrophobicity and rigidly forms a stable alpha-helix which mimics the structure of a fatty acid tail ^[2]. The aggregation of the ASIP domain would allow the formation of a micelle with a hydrophobic interior.

Fig 3: graphical illustration of the assembly process of this part

The domains were fused from the N terminus to C terminus following the order of “HRT2trunc-NT-ASIP”. A glycine-serine rich linker (red line in the diagram) was introduced between each domain to increase the flexibility in protein folding and adjusting the domains to correct positions. The final CDS of the designed protein was codon optimised for E. coli and synthesized onto a pET-28a(+) vector for in vitro characterizations.

Protein Structural Analysis

Fig 4: Protein structure of HRT2trunc-Lipo. The green/purple part is the HRT2trunc (deep blue), NT domain (light blue) and ASIP in yellow. The structure is compared with the HRT2trunc(green) in the left. Arrow shows the direction of growth of the rubber chain.

To understand the folding and the behavior of this fusion protein. The complete coding sequence of the HRT2-NT-ASIP was fed into the online alphafold-3 server by Google Deep Mind to predict the 3D structure [3]. The structure predicted was analyzed in PyMol where all three major domains have been folded correctly as expected. Furthermore, a structural alignment was conducted to compare the HRT2trunc (green) structure with same domain (deep blue) in the fusion protein, where no major deviation in the structure of the functioning unit is found, and the flexibility of the His-tag (purple) is also within the acceptable range.

Fig. 5: RMSD plot of the molecular dynamic simulation. Most of the residues are vibrating in range of 1 Å. High RMSD means strong flexibility. High flexibility region identified around amino acid #140, around #190 and #330, corresponding to the His-tag, GS-linker1 and GS-linker2 respectively.

The predicted structure was further analyzed with molecular dynamic simulation [4]. The high flexibility of the Gly-Ser linker has then been spotted from the global RMSD plot. Most of the residues vibrate in range of 1Å, showing high overall protein stability.

Characterization

Protein Expression

In vitro characterization of this basic part is carried out using E. coli expression system. Where the CDS was placed on the pET-28a(+) vector backbone for IPTG induced gene expression and transformed to E. coli strain BL21(DE3) for characterization. Production of such protein is readily visible in the SDS-PAGE detailed below.

Fig.6: MOPS-Tris-Gly-SDS-PAGE profile of all BL21 transformants with different plasmids, induced overnight with 0.5mM IPTG at 25C after reaching OD =0.5. Lane L: protein precision plus dual color ladder marker Bio-rad. Lane 8: Cell lysate after induced expression of HRT2trunc-NT-ASIP.

The expression of the part has been confirmed by the MOPS-Tris-Gly-PAGE. Where a band is identified around 41kDa, indicated with a red arrow in the lane 7 and 8 (lysate supernatant and pellet from the induced cell culture).

Organelle Formation

We have investigated the formation of our artificial micelle using this part. This has been performed using a BODIPY staining technique, which the BODIPY molecule stains specifically with intracellular hydrophobic pockets via interactions with aliphatic polymers such as triglycerides and polyisoprene (rubber) [5][6].

Fig.7: The fluorescent reading with excitation at 485nm and emission at 520nm for the cells stained with BODIPY. pET-28a(+) (pET) transformed BL21 induced with both 0mM and 1mM IPTG at OD600 = 0.5 serves as the global negative control (grey). The transformants of pET28a_HRT2trunc-NT-ASIP (ASIP) is induced with same IPTG conditions, where all cells are then cultured at 30oC for 16hr. The experiment is carried with 5 repeats of each sample, where the max and min from each group are represented by the error bars. The mean is shown with a cross, and the medium is identified as with the line inside the box. The box represents the interquartile range of the data distribution.

After rinsing the strained cells 3 times with PBSG (5% glycerol in PBS) to remove excess BODIPY molecule inside the cell membrane, the fluorescence of each group of cells were measured [7]. It was then observed that the HRT2trunc-NT-ASIP expressing cell (ASIP 1mM IPTG) produces significantly more fluorescence comparing to the non-induced transformant and negative controls. A one-tailed student t test was hence conducted to further compare the induced and non-induced groups, where solid statistical evidence was given with p<0.001 to further justify this observation. The average fluorescence of the induced cell is over 158% higher than the cells with no inducer added.

To further evident that our HRT2trunc-NT-ASIP is forming an artificial organelle that holds hydrophobic rubber molecule. We then imaged the BODIPY stained cells under a confocal microscope to identify the location of the stains inside the bacteria.

Fig.8 (Left) Positive group, HRT2trun-NT-ASIP with IPTG induced; (Right) Negative group, without being IPTG induced. Notice the yellow signal in the central of individual cells of IPTG induced sample. The samples were observed at 100x magnification.

The confocal microscopy images have revealed significantly higher and more nucleated fluorescence signal inside the HRT2trun-NT-ASIP expressing E. coli. Whereby the Z-stack images which scanned through the E. coli cell have evidenced that the signal cluster is present inside the cell rather attaching to the cell membrane. This visually evidenced that the ASIP is mediating the formation of artificial hydrophobic chamber inside the E. coli after translated. Since BODIPY selectively dyes hydrocarbons, it is then highly evidence that these organelles are very likely to carry natural rubber [8].

Rubber production

The part was also characterized for its ability to produce natural rubber. The E. coli expressing this part is cultured in large quantity (100ml) and induced overnight with 1mM IPTG at 30C with a pET negative control after reaching OD600 = 0.5. The cells were lysed through the chemical method (detailed at Imperial_college_2024 wiki, contribution page), where rubber is extracted using cyclohexane [9]. The absorbance at the UV spectrum was then measured for the solutions.

Fig.8: Fig.9 (A): UV-vis absorbance curve of rubber extracted from the overnight cultures with cyclohexane. pET serves as a negative control. (B): comparing the absorbance value acquired at 210nm of different samples that contain natural rubber (cis-1,4-polyisoprene), ASIP is the HRT2trunc-NT-ASIP parts addressed in this registry.

The curve for ASIP is observed to be overall higher than the control group. Indicating a higher amount of polyisoprene being extracted with cyclohexane. The extract from cells expressing this part has also been compared with wild-type (pET28a) E. coli with known amounts of polyisoprene added. Through comparing the absorbance at 210nm a theoretical yield is thus determined to be around 0.1 to 0.5mg per 100ml of culture ^[10][11].

Burden

Fig 10: the growth curve conducted to study the burden of this part.

We have conducted a series of growth assays for this part to illustrate the burden to the cells. A minor burden is identified after 1mM IPTG induction, as the growth of the E. coli transformant is slightly hindered, but likely due to the high concentration of IPTG.

How to use this part for your iGEM project?

An unused part is not a good part. - Edward Jixiao Wu

We have here reported the characterized design of our fusion protein HRT2-NT-ASIP, which can form an artificial organelle through spidroin-inspired micelle formation. We have furthermore used the advance lipid-specific staining to showcase the hydrophobic interior of this organelle to be formed inside the E. coli and exemplified the use of this organelle as an environment to producing long aliphatic chains of natural rubber. However, we do realize the vast untapped potential of this part in future iGEM projects. To make it easier for future iGEMers to further develop from, we have documented a list of cloning tips that makes our part a modular design for the future iGEM teams.