Part:BBa_K4846018

pETduet-DarE-BsjM

pETduet-DarE-BsjM

Composite Part: BBa_K4846018

pETduet-DarE-BsjM

Usage and Biology

DarA is the core sequence of darobactin that requires modification by the post-translational modifying (PTM) enzyme DarE to generate the mature product. DarL is the sequence recognized and bound by DarE for modification[1]. BsjA is the amino acid sequence of bicereucin that requires modification by the PTM enzyme BsjM to form the mature product. BsjL is the sequence recognized and bound by BsjM for modification[2].

Construction Design

We constructed pETduet-DarE-BsjM and pRSFduet-DarL-BsjL-His-DarA-BsjA(BBa_K4846020) by enzymatic digestion and ligation, then transformed into the same strain. We induced expression in the positive transformant, then lysed the cells by sonication to release contents and proteins. To obtain the target fusion peptides at higher purity, we performed nickel column purification on the lysate supernatant. The purified proteins were cleaved in vitro by lysyl endopeptidase to release the core peptides.

Figure 1.Profile of pETduet-DarE-BsjM

Experimental Approach

We used the restriction enzymes to create complementary sticky ends on the vector (pETduet) and fragments (DarE-BsjM). T4 DNA ligase was then used to ligate the vector with the fragments, generating plasmid pETduet-DarE-BsjM.

Figure 2 The generation of the plasmids constructed.

After constructing the recombinant plasmid pETduet-DarE-BsjM, which was transformed into BL21(DE3) competent cell. We performed gel electrophoresis and sequencing to verify the successful transformation into E. coli.

Figure 3 Colony PCR and sequencing results of transformants containing plasmid pETduet-DarE-BsjM

Characterization/Measurement

We inoculated positive clones and induced protein expression with IPTG. After induction, bacterial cells were lysed by sonication to release cellular contents and proteins. Nickel column purification was then performed on the lysate supernatant to obtain the target fusion peptides at higher purity (Figure 4).

Figure 4 SDS-PAGE result of the protein expression and purification.

References:

1. Imai, Y., Meyer, K. J., Iinishi, A., Favre-Godal, Q., Green, R., Manuse, S., Caboni, M., Mori, M., Niles, S., Ghiglieri, M., Honrao, C., Ma, X., Guo, J. J., Makriyannis, A., Linares-Otoya, L., Böhringer, N., Wuisan, Z. G., Kaur, H., Wu, R., Mateus, A., … Lewis, K. (2019). A new antibiotic selectively kills Gram-negative pathogens. Nature, 576(7787), 459–464.
2. Huo, L., & van der Donk, W. A. (2016). Discovery and Characterization of Bicereucin, an Unusual d-Amino Acid-Containing Mixed Two-Component Lantibiotic. Journal of the American Chemical Society, 138(16), 5254–5257.

Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 149
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 305
  Illegal BglII site found at 7506
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 324
  Illegal NgoMIV site found at 671
  Illegal NgoMIV site found at 5348
  Illegal AgeI site found at 6666
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1256
  Illegal BsaI.rc site found at 8736
  Illegal SapI.rc site found at 2916