Difference between revisions of "Part:BBa K525517"

Revision as of 13:36, 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
Degradation of BPA	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]

@@ Line 6: / Line 6: @@
 ===Usage and Biology===
-Expressing this BioBrick in ''E. coli'' enables the bacterium to degrade the endocrine disruptor bisphenol A (BPA).
+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>)''']]
 ===Important parameters===