View source for Part:BBa K3332028


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<partinfo>BBa_K3332028 short</partinfo>

Aminoalkylphosphonate N-acetyltransferase with a 6*histidine-tag, which can degradate AMPA. Use <partinfo>BBa_K823004</partinfo> to construct a new part that can catalyze the acyl transfer reaction between aminomethylphosphoric acid and acetyl-CoA, his tag is used for protein purification.


===Biology===

Under natural conditions, many microorganisms can use the glyphosate oxidoreductase to oxidize glyphosate to aminomethylphosphoric acid (AMPA). 

Phn system is a gene cluster for organophosphorus transport and degradation in many microorganisms. The phnO gene encodes the acyltransferase which catalyzes the transfer of acyl to AMPA by acetyl-coa. Through this one-step acyl transfer reaction, AMPA can be converted into glyphosate analogue, which can then be degraded by C-P lyase.


===Usage===

In order to easily purify PhnO, we added a his-tag (6*His) at the C-terminal. we ligased the strong promoter (<partinfo>BBa_J23100</partinfo>) and the parts (RBS-phnO-Terminator) on the expression vector pSB1C3 by standard assembly. Then the ligation mixture was transformed into ''E. coli'' DH5α & ''E. coli'' BL21 (DE3), and the correct recombinant one was confirmed by chloramphenicol, enzyme-cut identification and sequencing.


===Characterization===

'''1. Agarose Gel Electrophoresis'''

When we were building this circuit, enzyme-cut identification was used to certify the plasmid was correct. We used the ''Eco''R I and ''Pst'' I to cut the plasmid, then we got the target separate fragment-686bp.
<table><tr><th>[[File:T--XMU-China2020--BBa K3332028 1.png|thumb|300px|Fig.1 The result of plasmid cut with enzyme ''EcoR''I and ''Pst''I. Plasmid: pSB1C3.]]</th><th></table>

'''2. SDS-PAGE'''

The constructed plasmid was transformed into ''E. coli'' BL21 (DE3). Owing to the his-tag, the PhnO could be easily purified through AKTA with Ni-NTA. Compared with the lysate, the PhnO in the eluant can be found at around 16 kDa. Both of them were electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining.
<table><tr><th>[[File:T--XMU-China2020--BBa K3332028 2.png|thumb|300px|Fig.2 SDS-PAGE analysis of protein in lysate of ''E. coli'' BL21 (DE3) cells and the eluant after AKTA by Coomassie blue staining. Target bands can be seen at about 16 kDa]]</th><th></table>

'''3. Determination of enzyme kinetic constants'''

Velocities for the reaction of PhnO were determined using 4,4′-dithiodipyridine (DTDP) to continuously detect the formation of the product CoA at 324 nm (thiopyridone: ε= 19800 M<sup>-1</sup> ·cm<sup>-1</sup> ) at 25 °C. (1) Enzyme activity was determined through enzyme-labeled instrument.

Km and kcat of different substrates were determined with a concentration gradient.
<table><tr><th>[[File:T--XMU-China2020--BBa K3332028 6.png|thumb|500px|Fig 3. The relationship of 1/Enzyme activity and 1/concentration of AcCoA and AMPA. Used to determine the enzyme kinetic constants.]]</th><th></table>
<table><tr><th>[[File:T--XMU-China2020--BBa K3332028 8.png|thumb|300px|Table 1. Enzyme kinetic constants with two substrates.]]</th><th></table>
<ref>1.	J. C. Errey, J. S. Blanchard, Functional Annotation and Kinetic Characterization of PhnO from Salmonella enterica. Biochemistry 45, 3033-3039 (2006).</ref>

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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K3332028 SequenceAndFeatures</partinfo>


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===Functional Parameters===
<partinfo>BBa_K3332028 parameters</partinfo>
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