Difference between revisions of "Part:BBa K525517"

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<partinfo>BBa_K525517 parameters</partinfo>
 
<partinfo>BBa_K525517 parameters</partinfo>
 
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===References===
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<biblio>
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#Sasaki pmid=18492046
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#Sasaki05a pmid=16332782
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</biblio>

Revision as of 13:37, 17 September 2011

Fusion protein of BisdA and BisdB behind constitutive promoter

Fusion protein of BisdA and BisdB behind constitutive promoter.


Usage and Biology

Expressing this BioBrick in E. coli enables the bacterium to degrade the endocrine disruptor bisphenol A (BPA). BPA is mainly hydroxylated into the products 1,2-Bis(4-hydroxyphenyl)-2-propanol and 2,2-Bis(4-hydroxyphenyl)-1-propanol. In S. bisphenolicum AO1, a total of three genes are responsible for this BPA hydroxylation: a cytochrome P450 (CYP, bisdB), a ferredoxin (Fd, bisdA) and a ferredoxin-NAD+ oxidoreductase (FNR) Sasaki05a. The three gene products act together to reduce BPA while oxidizing NADH + H+. The cytochrome P450 (BisdB) reduces the BPA and is oxidized during this reaction. BisdB in its oxidized status is reduced by the ferredoxin (BisdA) so it can reduce BPA again. The oxidized BisdA is reduced by a ferredoxin-NAD+ oxidoreductase consuming NADH + H+ so the BPA degradation can continue Sasaki05a. This electron transport chain between the three enzymes involved in BPA degradation and the BioBricks needed to enable this reaction in vivo and in vitro are shown in the following figure (please have some patience, it's an animated .gif file):

Fig. 1: Animation of proposed reaction mechanism of bisphenol A hydroxylation by the involved enzymes FNR (BBa_K525499, Fd (BisdA, BBa_K123000) and CYP (BisdB, BBa_K123001)

Important parameters

Experiment Characteristic Result
Expression in E. coli Compatibility E. coli KRX, TOP10, MACH1, BL21(DE3)
Expression Constitutive
Optimal temperature 30 °C
Working BPA concentration 120 mg L-1 (0.53 mM)
Purification Molecular weight 59.3 kDa
Theoretical pI 4.99
High absorbation 450 nm
Degradation of BPA Completely degradation of 0.53 mM BPA 21 - 24 h
Specific BPA degradation rate 1.29 10-10 mM cell-1

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 664
    Illegal BamHI site found at 1402
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 65
    Illegal AgeI site found at 1655
  • 1000
    COMPATIBLE WITH RFC[1000]



References

<biblio>

  1. Sasaki pmid=18492046
  2. Sasaki05a pmid=16332782

</biblio>