Part:BBa_K1228009

38aa and 25aa fusion peptide with T1 terminator and Constitutive promoter veg

Human lysozyme (hLZ), the major protein in breast milk, exhibits microbicidal activity to various degrees against several bacterial strains. The HLH peptide(38Aminoacid peptides) and its N-terminal helix (H1) were the most potent bactericidal to bacteria. Evidence shows that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and is able to dissipate the membrane-potential-dependent respiration of Gram-positive bacteria. Lactoferrin (Lf) is a multifunctional member of the transferrin. Lf is found at the mucosal surface where it functions as a prominent component of the first line of the host defense system against infection and inflammation. The protein(25Aminoacid peptides) is part of Lf.it has a broad spectrum of anti-infection and anti-inflammatory function. Also, lactoferrin has a broad anti-bacterial range and strong anti-bacterial activity. 25 aminoacid peptides and 38 aminoacid peptides are combined with Promoter veg(https://parts.igem.org/Part:BBa_K541503) and T1 terminator. This fusion protein may have much more strong anti-bacterial activity.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Experiment and Results

1. Inoculate recombinant strains into 20ml LB.Cultivate at 37°C overnight.

2. Add 200ul indicator inoculum (Micrococcus Lysodeikticus or Escherichia Coli) untill heated LB solid medium (100ml) cool to about 50°C.

3. Use puncher make several hole on the LB plate.Add 80ul recombinant strains inoculum into the hole.Cultivate at 37°C overnight.

4. Observe whether LB plate will appear inhibition zone.

Reference

1.BellamyW,TakaseM,Yamauchi K,et al. Identification of the bactericidal domainof lactoferrin[J]. BiochimicaetBiophysicaActa,1992,1121( 1/2):130-136.

2.EliassenLT,Berge G,SveinbjornssonB,et al. Evidenceforadirect antitumormechanismof actionof Bovinel actoferricin[J].AnticancerResearch,2002,22(5):2703－2710.

3.Wakabayashi H,TakaseM,TomitaM. Lactoferricin derived from milk protein lactoferrin[J]. Current Pharmaceutical Design,2003,9(16):1277－1287.

4.Hisham R. Ibrahim,* Kenta Imazato, and Hajime Ono. Human Lysozyme Possesses Novel Antimicrobial Peptides within Its N-terminal Domain that Target Bacterial Respiration. J Agric Food Chem. 2011 Sep 28;59(18):10336-45. doi: 10.1021/jf2020396. Epub 2011 Sep 2.