Difference between revisions of "Part:BBa K5416062"
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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. | 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. | ||
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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. | 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. | ||
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Revision as of 12:28, 30 September 2024
pT7LacO + P22 + HRT2-SP + T7term
This is a composite part consisting of two CDS: P22 and HRT2-SP protein, placed downstream of pT7-LacO promoter (BBa_K2406020) for inducible overexpression. P22-His (BBa_K5416061) is the bacteriophage P22 mature virion capsid protein fused with a His-tag. The HRT2-SP encodes a truncated cis-prentyltransferase HRT2trunc(BBa_K5416000) derived from H. brasiliensis that is recombined with a scaffold-protein (SP, BBa_K3187021) 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, created with Biorender
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
Fig 3: Assembly illustration diagram of this composite part
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.
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.
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.
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.
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.
Burden
Fig.9 The growth curve of E. coli cells expressing this part comparing to non-induced control groups
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
- 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.
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. :)
The first RBS sequence:
AAATA ATTTT GTTTA ACTTT AAGAA GGAGA TATAC
The sequence in brown (html:#800000) is the anti-rRNA region where provides anchorage of the RBS to the mRNA. Flanking sequence are designed according to the rest of the mRNA to reduce unwanted mRNA structures.
The second RBS sequence
ATCCA ATTCT AAACA CATAA GGAGG TAATA T
The sequence in brown (html:#800000) is the anti-rRNA region where provides anchorage of the RBS to the mRNA. Flanking sequence are designed according to the rest of the mRNA to reduce unwanted mRNA structures.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 636
Illegal AgeI site found at 1037 - 1000COMPATIBLE WITH RFC[1000]