Difference between revisions of "Part:BBa K3332028"

Revision as of 18:42, 27 October 2020

phnO-his-tag

Aminoalkylphosphonate N-acetyltransferase with a 6*histidine-tag, which can degradate AMPA. Use BBa_K823004 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 (BBa_J23100) 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 EcoR I and Pst I to cut the plasmid, then we got the target separate fragment-686bp.

Fig.1 The result of plasmid cut with enzyme EcoRI and PstI. Plasmid: pSB1C3.

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.

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

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-1·cm-1) at 25 °C. (1) Enzyme activity was determined through enzyme-labeled instrument.

Km and kcat of different substrates were determined with a concentration gradient.

Fig 3. The relationship of 1/Enzyme activity and 1/concentration of AcCoA and AMPA. Used to determine the enzyme kinetic constants.

Table 1. Enzyme kinetic constants with two substrates.

^[1]

Sequence and Features

1. ↑ 1. J. C. Errey, J. S. Blanchard, Functional Annotation and Kinetic Characterization of PhnO from Salmonella enterica. Biochemistry 45, 3033-3039 (2006).