Part:BBa_K3332072

J23100-RBS-phnO-his-tag-terminator

        Aminoalkylphosphonate N-acetyltransferase with a 6*histidine-tag, which can degradate AMPA. We use BBa_K823004-BBa_B0034 to construct a new part that can improve the capability of chassis bacteria to degrade glyphosate.

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 transportation and degradation in many microorganisms. The phnO gene encodes the acyltransferase which catalyzes the transfation of acyl to AMPA by acetyl-coa. Through this one-step acyl transfer reaction, AMPA can be converted into glyphosate analogue, which can be degraded by C-P lyase later.

Usage

        In order to purify PhnO easily, we added a his-tag (6*His) at the C-terminal. we ligased the strong promoter、RBS、Terminator(BBa_J23100、BBa_B0034、BBa_B0015) and the parts (phnO) 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

         We use restriction enzyme to digest this circuit with EcoR I and Pst I to cut the plasmid to certify that the plasmid was correct. Then we got the target separate fragment-686bp.

Fig.1 The result of plasmid cut with enzyme EcoR I and Pst I. 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.

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 220
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]