Part:BBa_K4195114

I0500-B0034-his-linker-edl060-B0015

His-tag enable us to purify the linking protein. This part is used to verify the killing function of endolysin Lysqdvp001 encoded by edl060 gene on E. coli BL21(DE3), Vibrio natriegens VnDX and Vibrio alginolyticus ATCC33787.In order to make bond with nickel column to purify Lysqdvp001, we added a his-tag (6*His) at the N-terminal of Lysqdvp001. And to reduce the possibility that the his-tag may affect the function of Lysqdvp001, we added a linker between his-tag and Lysqdvp001.

Usage

We used both BBa_I0500 and BBa_B0015 to construct the expression system and obtained this circuit, which are assembled on the expression vector pUC57-Simple by standard assembly. The constructed plasmids were transformed into E. coli DH5α and BL21(DE3), then the positive colonies were selected by ampicillin and confirmed by colony PCR and sequencing. The positive colonies were cultivated and induced by L-arabinose, and GE AKTA Prime Plus FPLC System was employed to get purified protein. Then the purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. The purified protein underwent dialysis, freeze-drying and re-dissolution, and was employed to validate the killing effect on E. coli BL21(DE3), Vibrio natriegens VnDX and Vibrio alginolyticus ATCC33787 which were treated with EDTA.

Biology

Edl060 Edl060 is a gene encoding enzyme called endolysin Lysqdvp001 from bacteriophage qdvp001 with specificity for Vibrio parahaemolyticus that degrade the peptidoglycan cell wall of Vibrio parahaemolyticus (1).

Characterization

Identification

Agarose Gel Electrophoresis When we were constructing this circuit, regular PCR was used to certify the plasmid was correct. We got the target bands(2193 bp) at the position between 2000bp and 3000bp.

Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195114_pUC57-Simple. Target bands can be observed at the position between 2000 bp and 3000 bp.

SDS-PAGE

The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by L-arabinose, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image of his-linker-edl060 tag (Fig. 2), the target protein (26.0 kDa) could be observed at the position between 25 kDa and 30kDa on the purified protein lanes (FR).

Fig. 2 SDS-PAGE analysis of protein in lysate of E. coli BL21(DE3) and the elution samples. Target bands (26.0 kDa) can be observed at the position between 25 kDa and 30kDa

Killing effect verification

To proof the killing specificity of Lysqdvp001, purified enzyme was tested on E. coli BL21(DE3), Vibrio natriegens VnDX and Vibrio alginolyticus ATCC33787 which had been treated with EDTA. And then the mix was applied to agar spot test.

Fig. 3 The result of killing effect verification showing on plates. The first line is results with the EDTA+Sterilized water treated. The second line is results with the EDTA+Lysqdvp001 treated. The third line is results with Alkaline Lysis treated. Every line can be divided into groups inlude E.coli group, Vibrio alginolyticus, Vibrio natriegens. The first column is groups that bacteria been diluted 10³ times. The second column is groups that bacteria been diluted 10⁴ times. The third column is groups that bacteria been diluted 10⁵ times. The forth column is groups that bacteria been diluted 10⁶ times. By contrast, it shows that Lysqdvp001 has killing effect on Vibrio alginolyticus, but not on E.coli and Vibrio natriegens.

Reference

1. Wang W, Li M, Lin H, Wang J, Mao X. The Vibrio parahaemolyticus-infecting bacteriophage qdvp001: genome sequence and endolysin with a modular structure. Arch Virol. 161(10):2645-52.(2016).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961