Difference between revisions of "Part:BBa K5416030"
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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. | 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. |
Latest revision as of 08:46, 1 October 2024
PhaP PhaC-HRT2trunc
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]
Fig 2: An illustration of how this part is designed to work, created with Biorender
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).
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
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]