Part:BBa_K4613025

pQE-80L-C3

The composite part was constructed to find an appropriate expression intensity to achieve balance between metabolic burden and detection efficiency. Moreover, we hope our C3, which can be found under https://parts.igem.org/Part:BBa_K4613012, can be expressed successfully in E. coli Nissle 1917 (EcN). We tried T5 lac promoter from pQE-80L.

However, the amount of its protein expression is depressing.

Fig. 1 Results of pQE-80L-C3. (a) The plasmid map of pQE-80L-C3. (b) SDS-PAGE analysis of protein expression trials in E. coli BL21(DE3) cultured in Terrific Broth medium overnight using pQE-80L-C3. The temperature was 20℃. Lane M: protein marker. Lane 1: induced total protein. Lane 2: precipitation. Lane 3: supernatant. c. SDS-PAGE analysis of protein expression trials in E. coli BL21 (DE3) cultured in Terrific Broth medium for 4 hours using pQE-80L-C3. The temperature was 37℃. Lane M: protein marker. Lane 1: induced total protein. Lane 2: precipitation. Lane 3: supernatant.

Reference

1. Dai Z, Yang X, Wu F, et al.Living fabrication of functional semi-interpenetrating polymeric materials[J].Nat Commun,2021, 12 (1): 3422.
2. Zakeri B, Fierer J O, Celik E, et al.Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin[J].Proc Natl Acad Sci U S A,2012, 109 (12): E690-7.
3. Reddington S C, Howarth M.Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher[J].Curr Opin Chem Biol,2015, 29: 94-9.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1081
  Illegal BamHI site found at 1026
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1191
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 94