Difference between revisions of "Part:BBa K525562"

Revision as of 16:41, 28 October 2011

Fusion protein of NADP+ Oxidoreductase and BisdA and BisdB with middle strong promoter,RBS

Fusion protein of ferredoxin-NADP+ oxidoreductase, BisdA and BisdB; RFC 25 (Freiburg BioBrick assembly standard) for characterization of intra- and extracellular BPA degradation.

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)

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 1411
Illegal BamHI site found at 2149
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 65
Illegal AgeI site found at 2402
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 746

@@ Line 3: / Line 3: @@
 Fusion protein of ferredoxin-NADP+ oxidoreductase, BisdA and BisdB; RFC 25 (Freiburg BioBrick assembly standard) for characterization of intra- and extracellular BPA degradation.
+===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<sup>+</sup> oxidoreductase (FNR) <cite>Sasaki05a</cite>. The three gene products act together to reduce BPA while oxidizing NADH + H<sup>+</sup>. 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<sup>+</sup> oxidoreductase consuming NADH + H<sup>+</sup> so the BPA degradation can continue <cite>Sasaki05a</cite>. 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):
+[[Image:Bielefeld-Germany2011-BPAdegrad2.gif|center|700px|thumb|'''Fig. 1: Animation of proposed reaction mechanism of bisphenol A hydroxylation by the involved enzymes FNR (<partinfo>K525499</partinfo>), Fd (BisdA, <partinfo>K123000</partinfo>) and CYP (BisdB, <partinfo>K123001</partinfo>)''']]
 <!-- Add more about the biology of this part here
@@ Line 16: / Line 24: @@
 <partinfo>BBa_K525562 parameters</partinfo>
 <!-- -->
-===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<sup>+</sup> oxidoreductase (FNR) <cite>Sasaki05a</cite>. The three gene products act together to reduce BPA while oxidizing NADH + H<sup>+</sup>. 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<sup>+</sup> oxidoreductase consuming NADH + H<sup>+</sup> so the BPA degradation can continue <cite>Sasaki05a</cite>. 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):
-[[Image:Bielefeld-Germany2011-BPAdegrad2.gif|center|700px|thumb|'''Fig. 1: Animation of proposed reaction mechanism of bisphenol A hydroxylation by the involved enzymes FNR (<partinfo>K525499</partinfo>), Fd (BisdA, <partinfo>K123000</partinfo>) and CYP (BisdB, <partinfo>K123001</partinfo>)''']]