Difference between revisions of "Part:BBa K1403019:Design"

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===Design Notes===
 
===Design Notes===
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This sequence was submitted downstream a standard T7 constitutive promoter inside a pETDUET vector.
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The purpose of this part is to overexpress <i>ilvB/N</i> in <i>E. coli</i>.
  
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The ilvBN sequence was isolated from MG1655 through PCR and digested with NCoI and EcoRI to put into pETDUET1 vector. It was transformed into E.Coli strain with plasmid containing aldB to produce acetoin, which has the smell of butter.
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The <i>ilvB/N</i> genes were isolated from <i>E. coli</i> MG1655 through PCR, digested with NcoI and EcoRI, and cloned into the pETDUET-1 vector downstream a T7 promoter and RBS.  
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This part is not strictly necessary for production of acetoin in <i>E. coli</i> but improves the production thereof.
  
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We chose pETDUET1, instead of PSB1C3, because this vector has promoter in itself and makes the cloning process easier. This part is not necessary for production of any acetoin, but important for a high amount.
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Oligos:
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Fwd: 5'- GTTCAAGCCTTGAGCGGTTACTG -3'
  
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===Source===
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Rev: 5'- GAACTTGCCATGCTCCAGTCCT -3'
  
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E. coli MG1655
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===Source===
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<i>ilvB/N</i> coding sequences from <i>E. coli</i> MG1655 ([http://www.ncbi.nlm.nih.gov/nuccore/545778205?from=3850802&to=3851092&sat=4&sat_key=117098803&report=gbwithparts U00096.3])
  
 
===References===
 
===References===
  
 
David R. Nielsen, Sang-Hwal Yoon, Clara J. Yuan and Kristala L. J. Prather, Metabolic engineering of acetoin and meso-2,3-butanediol biosynthesis in E. coli, January 2010.
 
David R. Nielsen, Sang-Hwal Yoon, Clara J. Yuan and Kristala L. J. Prather, Metabolic engineering of acetoin and meso-2,3-butanediol biosynthesis in E. coli, January 2010.

Latest revision as of 07:41, 31 October 2014

Acetohydroxybutanoate synthase /acetolactate synthase (ilvB/N) expression cassette


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 47
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The purpose of this part is to overexpress ilvB/N in E. coli.

The ilvB/N genes were isolated from E. coli MG1655 through PCR, digested with NcoI and EcoRI, and cloned into the pETDUET-1 vector downstream a T7 promoter and RBS. This part is not strictly necessary for production of acetoin in E. coli but improves the production thereof.

Oligos: 

Fwd: 5'- GTTCAAGCCTTGAGCGGTTACTG -3'

Rev: 5'- GAACTTGCCATGCTCCAGTCCT -3'

Source

ilvB/N coding sequences from E. coli MG1655 ([http://www.ncbi.nlm.nih.gov/nuccore/545778205?from=3850802&to=3851092&sat=4&sat_key=117098803&report=gbwithparts U00096.3])

References

David R. Nielsen, Sang-Hwal Yoon, Clara J. Yuan and Kristala L. J. Prather, Metabolic engineering of acetoin and meso-2,3-butanediol biosynthesis in E. coli, January 2010.