Revision as of 00:33, 29 September 2024

HRT2trunc-NT-ASIP

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.

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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.

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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 (<a href="https://parts.igem.org/Part:BBa_K1088003">BBa_K1088003</a>) 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 (<a href="https://parts.igem.org/Part:BBa_K3264000">BBa_K3264000</a>), 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.

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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.

To use our artifical organelle as a platform, simply replace the HRT2-trunc encoding sequence with your enzyme of interest.

Fig 11: the modifiable domains in this part

This diagram above gives a detailed annotation of all the features in this part. Where simply changing the HRT2trunc encoding sequence (base 1 to 561) with a protein of choice would allow the formation of an artifical organelle dispalying your protein of interest. It is worth to consider that the hydrophobicity of the protein on the outter surface could potentially impact the structural integrity of the organelle and using a hydrophobic protein would thus be advised. The second major domain NT and the second linker (base 580 to 999) severs important role of orienting the hydrophobic domain inward to form the micelle, this sequence should hence be conserved in most of the cases.

Fig 12: Proposed assembly strategy

We encourage the use of Gibson assembly to use this part. But we has still made it compatible with Biobricks and GoldenGate for people to use different methods.
As mentioned before, any protein of interest could be inserted between base 1 to 561 (or 580 if to discard the first linker). Where it would be neccessary to design the overhangs of primers according the targeting seqeunce and the assembly method used.

Fig 13: Future focuses

• Ratio between ASIP and attaching proteins: Project could focus on introducing extra source of NT-ASIP to aid the fomarion of the organelle. This could be done by designing a composite part where the base region 580 to 1140 of this part is placed downstream of an inducible promoter.
• Exploring ASIP library: In theory any hydrophobic helix could be used to produce this micelle, selecting a phase-separating domain which have different chemcial property may increase the micelle formation and allowing the encapsulation of different substance with different properties.
• Introducing Smart Release System: It would be a great success in engineering if a system is developed to release this organelle under control. An example of such system could be a UV-inducible suicide switch Part:BBa_K518010

References

1. Kronqvist N, Sarr M, Lindqvist A, Nordling K, Otikovs M, Venturi L, Pioselli B, Purhonen P, Landreh M, Biverstål H, Toleikis Z. Efficient protein production inspired by how spiders make silk. Nature communications. 2017 May 23;8(1):15504.
2. Nilsson, G. et al. Synthetic peptide-containing surfactants—evaluation of transmembrane versus amphipathic helices and surfactant protein C poly-valyl to poly-leucyl substitution. Eur. J. Biochem. 255, 116–124 (1998).
3. Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, Ronneberger O, Willmore L, Ballard AJ, Bambrick J, Bodenstein SW. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 2024 May 8:1-3
4. Kuriata A, Gierut AM, Oleniecki T, Ciemny MP, Kolinski A, Kurcinski M, Kmiecik S. CABS-flex 2.0: a web server for fast simulations of flexibility of protein structures. Nucleic acids research. 2018 Jul 2;46(W1):W338-43.
5. Yokota S, Gotoh T. Effects of rubber elongation factor and small rubber particle protein from rubber-producing plants on lipid metabolism in Saccharomyces cerevisiae. Journal of bioscience and bioengineering. 2019 Nov 1;128(5):585-92.
6. Govender T, Ramanna L, Rawat I, Bux F. BODIPY staining, an alternative to the Nile Red fluorescence method for the evaluation of intracellular lipids in microalgae. Bioresource technology. 2012 Jun 1;114:507-11.
7. Benito V, Goñi-de-Cerio F, Brettes P. BODIPY vital staining as a tool for flow cytometric monitoring of intracellular lipid accumulation in Nannochloropsis gaditana. Journal of applied phycology. 2015 Feb;27:233-41.
8. Li G, Li J, Otsuka Y, Zhang S, Takahashi M, Yamada K. A BODIPY-based fluorogenic probe for specific imaging of lipid droplets. Materials. 2020 Feb 3;13(3):677.
9. Salvucci ME, Coffelt TA, Cornish K. Improved methods for extraction and quantification of resin and rubber from guayule. Industrial Crops and Products. 2009 Jul 1;30(1):9-16.
10. Asawatreratanakul K, Zhang YW, Wititsuwannakul D, Wititsuwannakul R, Takahashi S, Rattanapittayaporn A, Koyama T. Molecular cloning, expression and characterization of cDNA encoding cis‐prenyltransferases from Hevea brasiliensis: a key factor participating in natural rubber biosynthesis. European Journal of Biochemistry. 2003 Dec;270(23):4671-80.
11. Abu Hassan A. Experimental and computational study of latex clearing protein LcpK30 for rubber degradation (Doctoral dissertation, University of Nottingham).

Index

MIPTHIAFIL DGNGRFAKKH KLPEGGGHKA GFLALLNVLT YCYELGVKYA TIYAFSIDNF RRKPHEVQYV MNLMLEKIEG MIMEESIINA YDICVRFVGN LKLLDEPLKT AADKIMRATA KNSKFVLLLA VCYTSTDEPH HHHHHPYINP YPDVLIRTSG ETRLSNYLLW QTTNCILYSP HALWPEISGS SGSHTTPWTN PGLAENFMNS FMQGLSSMPG FTASQLDDMS TIAQSMVQSI QSLAAQGRTS PNKLQALNMA FASSMAEIAA SEEGGGSLST KTSSIASAMS NAFLQTTGVV NQPFINEITQ LVSMFAQAGM NDVSAGNSIP SSPVHLKRLK LLLLLLLLIL GALLLGLLLI VVVVVVLIVV VIVGALLGL

Above is the amino acid sequence of this part. Domains are highlighted in different colors: HRT2trunc (deep green #003300); Intrinsic 6xHis-tag (pink #ff99cc); N terminus of spidroin (light blue #00ffff); ASIP hydrophobic helix (light yellow #ffff99)

Sequence and Features