Part:BBa_K5416030

PhaP PhaC-HRT2trunc

Imperial-College 2024

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

This composite part, with two CDS BBa_K208001 and BBa_K5416031 placed downstream of a pT7-LacO promoter BBa_K2406020, is designed to produce an artificial organelle of rubber particle inspired by the formation of polyhydroxyalkanoate (PHA) carboxysomes.

Background

Fig 1: How PhaC makes PHA granules ^[1][2]

Polyhydroxyalkanoate, is a hydrophobic polymer made in many prokaryotes, and recently becoming a famous material as it can be used as a bio-degradable plastic. Whilst the polymer is very hydrophobic in nature, it can be readily produced in large quantities in prokaryotic system, comparing to rubber. Insights into the structure of the enzyme producing the PHA, the PHA synthase (PhaC), reveals the existence of a hydrophobic domain close to the active site. This domain is shown to be involved in the granule formation of the PHA and with absence of this domain, little PHA is made in the cell. This appease to be similar to the issue observed with bioproduction of natural rubber, where losing anchorage of the molecule results in significant reduction of the total amount of rubber produced.

Fig 2: An illustration of how this part is designed to work, created with Biorender

Inspired by the structure of PhaC, we questioned if the mechanism could be applied to the formation of rubber granules. Where the HRT2trunc (BBa_K5416000) is redesigned to be attached to the N terminus hydrophobic PHA binding domain of PhaC1. This would hypothetically capture the newly formed rubber chain and phase-separate the rubber granule with the rest of the cell. By introducing the other PHA-associated protein (PhaP), this structure can be then stabilized and become an ideal environment for rubber synthesis.

Design

Protein Engineering

Too long didn’t read:? Here is the design workflow of how the basic part of this composite collection is designed.

Fig 3: Our engineering workflow

The first challenge is to combine the HRT2truc with the hydrophobic domain of the PhaC. In return we have redesigned the protein-protein interaction to make fusion protein PhaC-HRT2trunc. A bridge peptide is introduced to form a rigid structure where we can now orient the exit of polyisoprene from the HRT2trunc close to hydrophobic domain of PhaC. The detailed design of this part can be referred to this page.

Plasmid Design

This composite part can produce PhaP and PhaC-HRT2trunc at the same time from one mRNA transcript. In prokaryotes, this is achieved by introducing a ribosome binding site (RBS) sequence prior to each CDS. However, it is very frequently reported that long mRNA transcript may contain intrinsic cis hairpin structures that may block the access to conventional RBS sequences. Therefore, we here to report an in silico RBS optimization process we have implemented in this part to combat this problem.

Fig 4: The predicted RBS strength of this composite part, measured in translational activation units (ta, small = 10, high = 10^4).

Through using online designing tools, we have adjusted the translation activation of all the first start codon of the two CDS to high activation rates ^[3]. This optimization also normalized the translation rate hence the two basic parts will be expressed at the same rate.

The final optimized RBS along with the basic parts are then assembled through direct de novo DNA synthesis, in the following order:

Fig 5: The assembly illustration diagram.

Characterization

Expression

Fig 5: SDS-PAGE of E. coli carrying this part, induced under 0.5mM IPTG overnigth for 25C, lane 1 is the cell lysate after expression, where both proteins are identified with a red arrow at around 12 and 42kDa

After inducing the cell with this part by adding 0.5mM IPTG, an SDS-PAGE was carried out whereby the two basic parts PhaP and PhaC-HRT2trunc has been identified in lane 1.

Reference

1. Shively JM, Cannon GC, Heinhorst S, Fuerst JA, Bryant DA, Gantt E, Maupin-Furlow JA, Schüler D, Pfeifer F, Docampo R, Dahl C. Intracellular structures of prokaryotes: inclusions, compartments and assemblages. InEncyclopedia of Microbiology, Third Edition 2009 Jan 1 (pp. 404-424). Elsevier.
2. Obruca S, Sedlacek P, Slaninova E, Fritz I, Daffert C, Meixner K, Sedrlova Z, Koller M. Novel unexpected functions of PHA granules. Applied microbiology and biotechnology. 2020 Jun;104:4795-810.
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.

Index

Please review the index of part K5416031 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.

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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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]