Difference between revisions of "Part:BBa K4169029:Design"

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We added His tag on the end of tmd sequence.
 
We added His tag on the end of tmd sequence.
 
Then optimized trimethylamine dehydrogenase, mutated amino acid 344 form Val to Cys.The primers we designed for mutation are showned below:
 
Then optimized trimethylamine dehydrogenase, mutated amino acid 344 form Val to Cys.The primers we designed for mutation are showned below:
 +
 
V344C-F: GACGACATCCGTTGTTGTATCGGCTG
 
V344C-F: GACGACATCCGTTGTTGTATCGGCTG
 +
 
V344C-R: CAGCCGATACAACAACGGATGTCGTC
 
V344C-R: CAGCCGATACAACAACGGATGTCGTC
  
 
===Source===
 
===Source===
  
−
111
+
The original gene sequence of tmd is from The European Nucleotide Archive (ENA). Coding: CDX46666.1.
 +
 
 +
The link is here: https://www.ebi.ac.uk/ena/browser/view/CDX46666
 +
 
 +
And tmd gene is originally in Mesorhizobium sp. ORS 3359.
 +
 
 +
We mutated amino acid 344 form Val to Cys.
  
 
===References===
 
===References===
 +
 +
[1] Loechel C, Basran A, Basran J, et al. Using trimethylamine dehydrogenase in an enzyme linked amperometric electrode Part 1. Wild-type enzyme redox mediation[J]. Analyst, 2003, 128(2): 166-172.
 +
 +
[2] Scrutton N S, Sutcliffe M J. Trimethylamine dehydrogenase and electron transferring flavoprotein[J]. Enzyme-Catalyzed Electron and Radical Transfer, 2000: 145-181.

Latest revision as of 13:04, 12 October 2022


Mutated TMADH


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 388
    Illegal PstI site found at 183
    Illegal PstI site found at 1782
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 388
    Illegal PstI site found at 183
    Illegal PstI site found at 1782
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 388
    Illegal XhoI site found at 1717
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 388
    Illegal PstI site found at 183
    Illegal PstI site found at 1782
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 388
    Illegal PstI site found at 183
    Illegal PstI site found at 1782
    Illegal AgeI site found at 879
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

GenScript synthesised tmd plasmids and performed codon optimization. We added His tag on the end of tmd sequence. Then optimized trimethylamine dehydrogenase, mutated amino acid 344 form Val to Cys.The primers we designed for mutation are showned below:

V344C-F: GACGACATCCGTTGTTGTATCGGCTG

V344C-R: CAGCCGATACAACAACGGATGTCGTC

Source

The original gene sequence of tmd is from The European Nucleotide Archive (ENA). Coding: CDX46666.1.

The link is here: https://www.ebi.ac.uk/ena/browser/view/CDX46666

And tmd gene is originally in Mesorhizobium sp. ORS 3359.

We mutated amino acid 344 form Val to Cys.

References

[1] Loechel C, Basran A, Basran J, et al. Using trimethylamine dehydrogenase in an enzyme linked amperometric electrode Part 1. Wild-type enzyme redox mediation[J]. Analyst, 2003, 128(2): 166-172.

[2] Scrutton N S, Sutcliffe M J. Trimethylamine dehydrogenase and electron transferring flavoprotein[J]. Enzyme-Catalyzed Electron and Radical Transfer, 2000: 145-181.