Part:BBa_K4613302

pET-29a(+)-T3-M-CPA

In order to find an appropriate expression intensity to achieve balance between metabolic burden and detection efficiency, we tried the T7 lac promoter from pET-29a(+). The composite part can be directly imported into pET-29a(+) vector and express T3-M-CPA induced with IPTG.

Fig. 1 (a)The plasmid map of pET29a(+)-T3. (b)SDS-PAGE analysis of the purified protein T3 in E. coli BL21 (DE3) cultured in LB medium express protein for 12 hours at 20°C . Lane M: protein marker. Lanes 1-6: flow through and elution containing 10, 50, 50, 100, 100, 250, 250 mm imidazole, respectively.

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.
4. Xiong L, Peng M, Zhao M, et al.Truncated Expression of a Carboxypeptidase A from Bovine Improves Its Enzymatic Properties and Detoxification Efficiency of Ochratoxin A[J].Toxins (Basel),2020, 12 (11).

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 1062
  Illegal BamHI site found at 1007
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1172
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 75