Part:BBa_K5416060

pT7LacO + P22 + HRT2-SP + T7 term

Imperial-College 2024

This part is designed by Team Imperial_College in iGEM 2024. It is reported to form an artificial organelle which serves as an enclosed chamber for natrual rubber production.

P22 HRT2-SP (Rubber cis-prentyltransferase HRT2, virus-like particle combination)

This is a composite part consisting of two CDS: P22 and HRT2-SP protein, placed downstream of pT7-LacO promoter for inducible overexpression. P22 (BBa_K3187021) is the bacteriophage P22 mature virion capsid protein fused with a His-tag. The HRT2-SP encodes a truncated cis-prentyltransferase HRT2(BBa_K5416000) derived from H. brasiliensis that is recombined with a scaffold-protein (SP, BBa_K3187017) domain that can interact with the inner surface of the P22 capsid. We here to report this designed composite part to be capable of forming a virus-like-particle (VLP) as an artificial organelle with a compartmentalized environment for rubber production.

We have shown this part to have the following functions:

• Forming purifiable artifical organelle
• Encapsulating enzymes
• Producing hydrophibc internal environment
• Producing small amount of rubber

Design

Virus-like-particle (VLP) is a hollowed, polygonal particle formed from oligomerized virus capsid proteins. It was reported that, the bacterial phage P22, can produce solubilized VLPs where cargos can be encapsulated inside via simple protein-protein interactions (by attaching a scaffold protein to the inner surface of the VLP). Patterson et. al., 2012 demonstrated that enzymes encapsulated in this format gain access to cytosol substrates and maintain their functionality [1][2]. Where this property was explored in iGEM 2019 by Team:TU_Darmstadt.

Fig 1: An illustration of how this part works

In our design, P22 VLP capsid protein with a recombinant HRT2 protein (HRT2-SP) are co-expressed. We replaced the dimerization domain of HRT2 with the scaffold protein (SP) to mediate its interaction to be encapsulated by the VLP. We hypothesized that the enzyme’s encapsulation prevents membrane integration of the rubber chain produced, enabling rubber particle formation inside the VLP via hydrophobic interactions. This would hence make the translated product form this part an artificial organelle that produces rubber inside.

Circuit designing

Specific to designing of this composite part, we placed both the P22 capsid and the HRT2-SP protein under a T7-LacO promoter. To overcome the potential cis repression of ribosomal binding sites by forming unwanted RNA hairpin structure in long transcripts of mRNA, the sequences of the two ribosomal binding sites are re-designed using De Novo DNA online designing platform [3]. In which the software generates RBS sequences with a known degree of base-pairings according to the local sequences of the mRNA.

Fig 2: Plot of translational activation magnitudes (RBS strengths) through out the mRNA transcript of this part

The redesigned RBS sequences not only exhibit higher affinity (au = 2 × 104) to the anti-RBS sequences to that on E. coli ribosomes, but also normalized this affinity to a known amount. Through this in-silico design, we ensure that the P22 protein and the HRT2-SP protein are produced at 1:1 ratio at any transcriptional rates. This prevents over-saturation the P22 but also allows a good availability of HRT2-SP to be present for VLP integration. NOTE: controlling gene expression with multiple RBS only feasible for prokaryotic chassis. For eukaryotes, please considering introns/extrons.

Fig 3: Assembly illustration diagram of this composite part

The CDS and designed RBS sequences was then assembled in the following order to build this part onto a pET-28a(+) backbone for characterization in E. coli. Note that the codon optimization was carried prior to RBS designing.

Protein Struncture Analysis

Fig 4: protein structure alignment of the P22 capsid protein with the HRT2-SP, where the scaffold protein domain (pink) is well aligned with the original SP (cyan) reported by previous work.

To achieve the in vivo encapsulation, the dimerization of HRT2 is replaced by Scaffold protein (SP) mediate cargo-specific encapsulation of this enzyme inside the P22 VLP (BBa). The structure of HRT2-SP predicted by alphafold, has been aligned with the original scaffold protein in the P22 virus to understand its binding to the inner surface of the capsid [4][5].

Characterization

Protein Expression

We have not only aimed to obtain the evidence of protein production, but also the successful formation of the VLP that serves as an encapsulating component. For this purpose, we conducted a detailed SDS-PAGE assay to trace the P22 and HRT2-SP throughout the protein production and purification process.

Fig 5: SDSPAGE analysis result of IPTG-induced E. coli cell (BL21(DE3)) transformed with this part, cultured 16hr at 25oC. From left to right: lane L: Transgen 10-180kDa protein marker; lane 1: cell pellet after lysed with BugBuster with 0.2mg/ml lysozyme for 30min at room temperature; lane 2: lysate supernatant centrifuged at 3k rpm for 10min; lane 3: lysate supernatant centrifuged at 12k rpm for 10min; lane 4: cells sampled directly from the culture media; lane 5: 0.45um PDMV-filtered lysate (after centrifuged at 3k rpm); lane 6: first wash (with PBS) after protein loaded to Ni-NTA columns; lane 7: second wash (PBS with 2mM imidazole) of the Ni-NTA column; lane 8: 5ul of 20x concentrated elute (200mM imidazole); lane 9: 80x concentrated lysate supernatant, 5ul. All lanes except lanes 8 and 9 were loaded with 10ul of samples. The P22 capsid protein around 46kDa and HRT2trunc protein around 26kDa were identified with red arrows in the gel.

The gel above shows all the coding proteins in this composite part have been successfully expressed and identified in E. coli strain BL21 after 16hrs of 1mM IPTG induction at 25oC. Indicated by the bands around 46kDa and 26kDa for P22 and HRT2-SP respectively. Following the lysis of the cells via BugBuster solution with 0.2mg/ml lysozyme (Sigma-Aldrich, UK), the P22 and HRT2-SP persistently to exist in the liquid phase regardless of high-speed centrifugation (both in after spinning at 3k and 12k rpm). This indicates the high solubility of the VLP. The lysate was then passed through a 0.45um filter to remove insoluble proteins and cell debris and loaded into Ni-NTA column for His-tag specific protein purification. It was observed that minimal amount of targeted protein was existing in the flowthrough of washing steps (with PBS and low concentration of imidazole), which suggesting the binding of the VLP proteins to the nickel ions via the his-tag. This hypothesis was further evidenced by identifying good amount of VLP proteins after the column is washed with imidazole (lane 8). This elute is concentrated 20 times by volume using a 30kDa filter spin column(Merck, USA). Noticeably, even the HRT2-SP protein does not carry any His-tags and is smaller than threshold (<30kDa) of the spin column filter. The band for HRT2-SP has also been identified in the same lane, which provides strong evidence that the HRT2-SP could only be trapped inside the VLP to resist the washes.

VLP Electron Microscopy

We then asked if the VLPs are forming in spherical structures as we expected, and if the structure endured the purification process. To verify this question, a transmission electron microscopy is carried out (TEM) to image the entire VLP.

Fig 6: Negatively stained transmission electron microscopy (TEM) imaged with 80kV at 15k magnification for the VLP concentrated in PBS. Spherical VLPs have been identified in the sample (with red arrows) of approximately 50nm in diameter. Regions are zoomed out on the left side of the image where the bar corresponds to 50nm.

The concentrated VLP after purification was sent to our external contractor (Service Bio, China), where the TEM was then pictured. The image X, shows several spots of spherical objects approximately 50nm in diameter exhibiting the characteristics to be our VLP [6]. This shows solid evidence that our artificial organelles are forming.

Hydrophobic Body Staining by BODIPY

With solid evidence of VLP formation, we then investigated if rubber particle is present inside the VLP. This investigation is carried out via BODIPY staining, which this stain binds specifically to intracellular aliphatic compounds and has been thus used to stain lipid bodies and rubber particles in vivo [7][8]. In which we have compared the staining result of E. coli cells expressing this part with the wild-type stains (with empty backbone pET28a) and non-induced strains.

Fig 7: BODIPY staining of BL21 transformed with pET28a (pET) and this composite part (VLP), cells were induced with 0mM (-) and 1mM IPTG (+) for 16hrs at 25oC, respectively. Each group is carried out with five repeats where the fluorescence of the cells was measured with excitation at 485nm and emission at 520nm (green) to quantify the BODIPY inside the cell. pET28a transformants serves as a global control.

After staining and washing off excessive BODIPY molecules with PBSG (5% glycerol), the fluorescence of the cells was measured with the plate reader, with a significant increase in the VLP expressing strain compared to the pET28a control. Where a student t-test revealed a p value smaller than 0.05.

Rubber Production

Fig 8: Upper image: The UV-vis absorbance spectrum of the natrual rubber extracted from cyclohexane for E. coli expressing this part (6-blank), comparing to a negative control (pET-blank, pET28a transformant). Lower image: comparision of A210 reading of E. coli expressing this part with negative control (pET28a strain), and a known postive control which 10ug of polyisoprene is added to the negative control. All absorbance values are blanked with absorbance spectrum of cyclohexane.

The rubber producing ability of this part is assessed with 100ml of culture of the transformant, where the natural rubber produced by this part is extracted with cyclohexane following the reported protocol [9][10]. The characteristic absorbance at 210nm is compared with the wild-type BL21 (pET28a transformant), and known concentrations of cis-polyisoprene, that was determined to be approximately 10ug per 100ml.

Burden

Fig.9 The growth curve of E. coli cells expressing this part comparing to non-induced control groups

We have not yet spotted a significant cytotoxicity of this composite part to the E. coli cell. Yet the final plateau reaching with the part-expressing strain is slightly lower than the non-expressing one (OD600 = 0.8 comparing to 0.9).

How to Use This Part

Unused parts are not good parts. - Edward Jixiao Wu

In summary, we have reported the design of a composite part which is capable of producing a virus-like-particle and can be used to hold natural rubber. However, this VLP could be further used as a platform technology for other applications not limited to bioproduction and drug delivery. This organelle should be able to encapsulate any soluble protein. Here we thus provide with a detailed description of how to use this part and for future teams to modify protein domains at will.

Fig.10 Step 1 is to learn the function of this part

Before the part sequence is modified in your project, we advise you to consult to this document to learn more about the part's features. The P22 capsid protein should be identified as a region where modification should be avoided. As it is involved in the formation of the VLP. The following region (grey) on the other hand is more acceptable to any edits.

• The his-tag can be replaced with any small purification tags
• Rubber synthase enzyme domains can be replaced - remember the scaffold peptide should be kept to import the cargo to the VLP.

Fig.11 Clong your parts in! We encourages using Gibson Assembly though the part also works with BioBricks and Goldengate (Bsa1, Aar1) enzymes.

Here is the proposed cloning method to compile your enzyme of interest to this VLP platform. A simple method would be putting the SP protein at the C terminus of your enzyme. To do so, simply replace the region form the second RBS to the SP. An advise is to keep the adjecent sequence of the RBS2, as changing could not garantee the same level of translation activation. A stop codon should also be introduced at the end of SP domain, to terminates the protein expression.

References

1. Das, S., Zhao, L., Elofson, K. and Finn, M.G. (2020). Enzyme Stabilization by Virus-Like Particles. Biochemistry, 59(31), pp.2870–2881. doi: https://doi.org/10.1021/acs.biochem.0c00435.
2. Yang J, Zhang L, Zhang C, Lu Y. Exploration on the expression and assembly of virus-like particles. Biotechnology Notes. 2021 Jan 1;2:51-8.
3. Reis AC, Salis HM. An automated model test system for systematic development and improvement of gene expression models. ACS synthetic biology. 2020 Oct 15;9(11):3145-56.
4. Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: making protein folding accessible to all. Nature methods. 2022 Jun;19(6):679-82.
5. Bittrich S, Segura J, Duarte JM, Burley SK, Rose Y. RCSB protein data bank: Exploring protein 3D similarities via comprehensive structural alignments. Bioinformatics. 2024 Jun 13:btae370.
6. McCoy K, Selivanovitch E, Luque D, Lee B, Edwards E, Castón JR, Douglas T. Cargo retention inside P22 virus-like particles. Biomacromolecules. 2018 Aug 9;19(9):3738-46.
7. 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.
8. 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.
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.

Index

Please review the index of part K5416001; K5416061 for protein amino acid sequences and annotations. :)

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Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1354
  Illegal AgeI site found at 507
  Illegal AgeI site found at 1755
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 625