Difference between revisions of "Part:BBa K2286002"

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===DhaA31 Works in Tobacco Chassis===
 
===DhaA31 Works in Tobacco Chassis===
  
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For the plant expression system, this year we did codon-optimized it and introduced it into tobacco to explore its potential for stable work in plants. After obtaining positive plants, we first carried out reverse transcription test. The results show that DhaA31 can be stably transcribed in tobacco. Furthermore, we used tobacco leaf to extract crude enzyme solution for activity detection, and the results showed that DhaA31 was able to work effectively in tobacco.
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For the plant expression system, this year we did codon-optimized it and introduced it into tobacco to explore its potential for stable work in plants. After obtaining positive plants, we first carried out reverse transcription test. The results show that DhaA31 can be stably transcribed in tobacco. Furthermore, we used tobacco leaves to extract crude enzyme solution for activity detection, and the results showed that DhaA31 was able to work effectively in tobacco.
  
  

Revision as of 12:30, 1 November 2017

AOs-DhaA31: ascorbate oxidase signal peptide and haloalkane dehalogenase mutant

DahA31 is a multi-site mutant of haloalkane dehalogenase(DhaA), and its mutations are located in 135 (I-> F), 176 (C-> Y), 245 (V-> F), 246 (L- > I), 273 (Y-> F). Compared with DhaA, DhaA31’s activity to 1,2,3-trichloropropane (TCP) has been increased by 32 times[1]。 Because this enzyme is from Rhodococcus rhodochrous, the original sequence is suitable for expression in prokaryotic cells. However, this sequence is codon - optimized for petunia and is more suitable for plant system. In addition, the N-terminal of the enzyme fused an N-terminal ER-targeting signal peptide which is beneficial to stable protein expression and cell wall localization [2].

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]


Characterization

DhaA31 Works in Tobacco Chassis

For the plant expression system, this year we did codon-optimized it and introduced it into tobacco to explore its potential for stable work in plants. After obtaining positive plants, we first carried out reverse transcription test. The results show that DhaA31 can be stably transcribed in tobacco. Furthermore, we used tobacco leaves to extract crude enzyme solution for activity detection, and the results showed that DhaA31 was able to work effectively in tobacco.


Fig 1.RT-PCR of positive plants.
Fig 2.The amount of 2,3-DCP generated within four hours.Enzymatic reaction in 200mM Trs-SO4 at pH 8.5 and 37℃. The data were measured by Gas chromatography.

References

1.Pavlova, M., et al., Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate. Nat Chem Biol, 2009. 5(10): p. 727-33.

2.Nanasato, Y., et al., Biodegradation of gamma-hexachlorocyclohexane by transgenic hairy root cultures of Cucurbita moschata that accumulate recombinant bacterial LinA. Plant Cell Rep, 2016. 35(9): p. 1963-74.