Part:BBa_K2920727:Design

Effective AMP (Antimicrobial Peptide)

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]

Design Notes

JJ01 JJ01(Fig.1) is a antimicrobial peptide which is originally modify from MAP0403. As previous study indicated that although MAP0403 has antimicrobial potency, it has side effect- hemolytic which is not suitable for potential.According to the research, the high amphipathic alpha helical character may interact with membrane which include the properties of detergent and its analogies between both of its interface. In order to solve the side effect, JJ01 changed the amphipathic structure arrangement of MAP0403 to interfere the detergent-like structure as the first and foremost strategy.After identify the antimicrobial activity, JJ01 is found to inhibited E coli, S. aureus and P. aeruginosa at 5 μM, 5 μM and 10 μM, respectively. The minimal inhibition concentration (MIC90) of P. aeruginosa increased twice, but E coli and S. aureus still maintain at 5 μM. Fortunately, jj01 was proved to significant decrease the hemolytic activity by break down the amphipathic structure.In conclusion, JJ01 is an effective antimicrobiaol peptide which has potential to replace antibiotic. Fig.1. Helical wheel projection of JJ01 Fig.2. RP-HPLC chromatogram of the crude peptide JJ01 Fig.3. RP-HPLC chromatogram of the pure peptide JJ01 Fig.4. MALDI-TOF-MS spectrum of pure JJ01

geneic engineerng method

3.4. Procedure

1. Identify target gene

2. Design primer

3. Polymerase chain reaction

4. DNA purification

5. Extract target DNA from plastid

6. First transformation

7. Confirm fragment size of plastid

8. Sequencing

9. Second transformation

10. Protein expression

11. Identify purification conditions

12. Protein expression and purification

13. Antimicrobial activity analysis

Source

1.We got and invented the antimicrobial peptide from SPPS synthesis method at chemistry lab first, after that ,we used the genetic engineering method to produce it.

2.We used the backbone which is gotten from igem kit.

3.The biobrick was from igem kit and bio company.

4.The e.coi was gotten from genetic engineering lab.

References

[1] Greenfield NJ. Methods to estimate the conformation of proteins and polypeptides from circular dichroism data. Analytical biochemistry 1996;235:1-10. [2] Kondejewski LH, Jelokhani-Niaraki M, Farmer SW, Lix B, Kay CM, Sykes BD, et al. Dissociation of antimicrobial and hemolytic activities in cyclic peptide diastereomers by systematic alterations in amphipathicity. The Journal of biological chemistry 1999;274:13181-92. [3] Epand RF, Mowery BP, Lee SE, Stahl SS, Lehrer RI, Gellman SH, et al. Dual mechanism of bacterial lethality for a cationic sequence-random copolymer that mimics host-defense antimicrobial peptides. Journal of molecular biology 2008;379:38-50. [4] Lauterwein J, Bosch C, Brown LR, Wuthrich K. Physicochemical studies of the protein-lipid interactions in melittin-containing micelles. Biochimica et biophysica acta 1979;556:244-64. [5] Yeaman MR, Yount NY. Mechanisms of antimicrobial peptide action and resistance. Pharmacological reviews 2003;55:27-55. [6] Straus SK, Hancock RE. Mode of action of the new antibiotic for Gram-positive pathogens daptomycin: comparison with cationic antimicrobial peptides and lipopeptides. Biochimica et biophysica acta 2006;1758:1215-23. [7] Tatko CD, Waters ML. The geometry and efficacy of cation-pi interactions in a diagonal position of a designed beta-hairpin. Protein science : a publication of the Protein Society 2003;12:2443-52. [8] Li SC, Goto NK, Williams KA, Deber CM. Alpha-helical, but not beta-sheet, propensity of proline is determined by peptide environment. PNAS 1996;93:6676-81.