Part:BBa_K5201000

Characterisation of BBa_K5201001: HongKong-UCCKE

Document BBa_K5201000

Part registry

BBa_K5201000

Part type

Coding sequence

Short description

pmHAS*1-703 is a gene from Pasteurella Multocida, optimized for expression of class II hyaluronic acid synthase (HAS) in E. coli

Long description

Biology

pmHAS encodes hyaluronan synthase (HAS) from Pasteurella Multocida. pmHAS is a class II HAS enzyme, which is a peripheral membrane protein that can function without cell membrane.

Usage and design

Our recombinant plasmid consists of pmHAS and kfiD which is expressed under promoter (BBa_R0010). We hope that the pmHAS can produce HAS enzyme for synthesis of HA. HA is widely utilized by the cosmetic industry for its significant water absorption and retention properties. And therefore, we want to further implement it for agricultural use.

Standard curve of HA

The cetyltrimethylammonium bromide (CTAB) solution will react with HA to form precipitate, which absorbs 400 nm light. 150µL of standard HA solution in range of 0g/L to 0.1g/L, 350µL acetate acid, 1mL of 2.5g/L CTAB solution at room temperature for 5 min is added in a 96-well plate. The absorbance of the precipitate at 400 nm is measured using a plate reader. The standard curve of OD400 against HA concentration is plotted.

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Fig. 6 HA calibration

Production of HA

Culture in glucose medium

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Figs. 7, 8 OD₄₀₀ of transformed and untransformed bacteria supplied with glucose

Culture in glucose and glucosamine medium

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Figs. 9, 10 OD₄₀₀ of transformed and untransformed bacteria supplied with glucose and glucosamine

We expressed pmHAS in DH5a E. coli under a lac promoter and measured the amount of HA produced. In both glucose medium and glucose + glucosamine medium, we observed a consistent increase in HA absorbance suggesting that the HAS is functional and able to synthesize HA in E. coli. We also observed that the addition of glucosamine, one of the precursors of HA, can further increase the HA yield.

Source

Pasteurella Multocida

Design considerations

To facilitate cell-free systems in the future, the pmHAS gene is designed to produce residues 1-703 which are used to produce HAS. The residues 704-972 are used to connect to the cell membrane. This truncated form allows HAS to be produced without attaching to the cell membranes.

Sequence

gaattcgcggccgcttctagagatgcatcaccatcatcaccacaataccctgagtcaggccattaaggcctataatagcaatgattatcagctcgcattaaaattatttgaaaaaagcgcggagatttatggtcgtaaaatcgttgaatttcagatcacgaaatgcaaagaaaagctgagcgcgcatccgtcggtaaattctgcgcatttaagtgtgaacaaggaagaaaaagtgaatgtctgcgattcccctctcgatattgccactcagctgttgctgtcgaacgttaaaaaattggtcctttcagattcagaaaaaaataccttaaaaaataaatggaaattgctgacggagaaaaaaagcgagaatgctgaagtgcgcgctgtggccctggtgccgaaagacttcccgaaagacctggtgctggcgcctctacctgatcatgtgaacgattttacatggtacaaaaaacgtaagaagcgcctgggaatcaagcctgaacaccaacacgtcggactgtccattatcgtgaccaccttcaaccgcccggcgattttatccattaccttggcgtgtttagttaaccagaaaacgcactatccgtttgaggtcatcgtgaccgatgatggcagccaggaagacttgagcccgattatccgccaatatgaaaataaattagacatacgctacgtacgtcagaaagataacggctttcaagcatcggcggcacgaaacatggggctgcggctagccaaatatgactttattggcctgctggactgtgatatggcgcccaacccgctgtgggtccattcttatgtagccgagctgctggaagatgatgatctgaccattattgggccgcgtaaatacatagatacccagcacattgatccgaaagattttttaaataacgcgtcgttactggagagccttcctgaagttaagactaataactctgtcgcggccaaaggggaaggtacggttagtctggattggaggcttgaacagttcgaaaaaacagaaaatctgcgtctcagcgactccccctttcgcttctttgcagccggtaacgttgccttcgcgaaaaagtggctgaataaaagtggcttcttcgatgaagagttcaaccattggggcggtgaagatgttgaatttgggtaccgtctcttccggtatggcagctttttcaaaaccatcgacggcatcatggcgtatcaccaggagcccccaggcaaggagaacgagaccgatcgcgaagcgggcaaaaatattacgcttgatatcatgcgtgaaaaagttccatacatttatcgaaagctgctgccgattgaagacagccacattaatcgcgtgccgctggtgtcgatttacattccggcgtacaactgtgcgaattatatccaacgttgcgttgattcagcgttgaaccaaaccgtagtcgacctggaagtgtgcatctgtaatgatggtagtactgataatacattagaggtaatcaacaaactgtatggaaacaacccgcgtgtgcgcattatgtcgaaaccaaacggcggcattgcatctgcgagcaacgctgccgtgtcgtttgccaagggctactatattggccagctggacagtgacgactacttggaaccggatgccgtggaactatgcctgaaggaatttctgaaagataagacgctggcatgcgtgtacaccaccaaccgcaatgtaaatccagacggcagcctcattgcaaatggctacaattggccggaatttagccgcgaaaaactcacaactgcaatgatcgctcatcattttcgtatgttcactattcgcgcttggcacttgacggacggtttcaatgaaaaaatagaaaacgcagttgactacgatatgttccttaaactttcagaggtcggaaaatttaagcatctgaacaaaatctgttacaatcgcgttcttcacggtgataacacgtcaatcaaaaaactaggtattcagaaaaaaaaccattttgtggtagtgaatcaaagcctgaaccgtcagggtatcacctattacaactatgatgaatttgatgatcttgatgagtcaagaaagtacattttcaacaaaaccgccgagtatcaggaagaaattgatattctcaaggacatttactagtagcggccgctgcag

Assembly compatibility

RFC10

Reference

Sze, J. H., Brownlie, J. C., & Love, C. A. (2016). Biotechnological production of hyaluronic acid: a mini review. 3 Biotech, 6(1), 67. https://doi.org/10.1007/s13205-016-0379-9