Part:BBa_K1919002

Constitutive promotor J23106 and downstream CecropinXJ

Biobrick BBa_K1919002 is a composite part, consisting of constitutive promotor J23106, RBS, TrxA tag, 6xHis tag, thrombin site, S tag, enterkinase site and the coding sequence of antimicrobial peptide CecropinXJ.

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 BglII site found at 506
  Illegal BamHI site found at 549
  Illegal XhoI site found at 750
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Biology

Antimicrobial peptides (AMPs) are a group of peptides that play roles in the innate immune system to protect the host from invading pathogens [1]. AMPs have minimal toxicity and low sensitivity effects to the host [2], which means antimicrobial peptides have the potential to be used to replace antibiotics in the future. Thus, the detrimental effects of antibiotics overuse will be released.

Cecropins, a group of small AMPs mainly found in the hemolymph of insects, consist of 31 39 amino acid residues and have a broad spectrum, high heat stability and potent bacteriostatic activity [3-5]. CecropinXJ (Part BBa_K1919000) is a member of the Cecropin family, which was first cloned from the larvae of the Xinjiang silkworm (Bombyx mori). Previous researches have determined the complete amino acid sequence of this molecule [6]. It has been demonstrated that CecropinXJ could be expressed in eukaryotic expression system such as Pichia pastoris [7] or prokaryotic expression system such as E.coli [8]. What’s more, CecropinXJ exhibited to have various activities such as antibacterial activity against both Gram‑positive and Gram-negative bacteria, as well as antifungal activity [8]. These characteristics indicate that CecropinXJ is an ideal antimicrobial substance to be used to treat foot diseases caused by microbes.

Results

This part is constructed based on part BBa_K1919000 [1], which express cecropinXJ continuously downstream of constitutive promoter J23106 [2](Fig. 1)

Fig.1 Schematic structure of CecropinXJ with promotor J23106

The combination of cecropinXJ and constitutive promoters were determined by PCR. (Fig. 2) However, there is no clear band being detected while using SDS-PAGE and Western blot, which might due to the high level expression of AMP inhibited the growth of host itself.

Fig.2 The result of colony PCR confirmed the successful combination of CecropinXJ and constitutive promoters J23106

References

[1] Boman HG: Peptide antibiotics and their role in innate immunity. Annu Rev Immunol 13: 61-92, 1995.

[2] Devine DA and Hancock RE: Cationic peptides: distribution and mechanisms of resistance. Curr Pharm Des 8: 703-714, 2002.

[3] Boman HG, Wade D, Boman IA, Wåhlin B and Merrifield RB: Antibacterial and antimalarial properties of peptides that are cecropin-melittin hybrids. FEBS Lett 259: 103-106, 1989.

[4] Moore AJ, Devine DA and Bibby MC: Preliminary experimental anticancer activity of cecropins. Pept Res 7: 265-269, 1994.

[5] Hancock RE and Lehrer R: Cationic peptides: a new source of antibiotics. Trends Biotechnol 16: 82-88, 1998.

[6] Li JY, Zhang FC and Ma ZH: Prokaryotic expression of cecropin gene isolated from the silk worm Bombyx mori Xinjiang race and antibacterial activity of fusion cecropin. Acta Entomol Sin 47: 407-411, 2004 (In Chinese).

[7] Tang X, Wang H, Kelaimu R, Mao XF and Liu ZY: Molecular cloning, expression of cecropin-XJ gene from silkworm and antibacterial activity in Pichia pastoris. Biotechnology 21: 26-31, 2011 (In Chinese).

[8] Xia L, Zhang F, Liu Z, Ma J and Yang J: Expression and characterization of cecropinXJ, a bioactive antimicrobial peptide from Bombyx mori (Bombycidae, Lepidoptera) in Escherichia coli. Experimental and Therapeutic Medicine 5: 1745-1751, 2013.