Part:BBa_K2278021

D-NY15 Antimicrobial peptide with Alpha-Factor Secretion Signal

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 244
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

This DNA biobrick was designed in order to produce in strain.

1- Biological background

Mécanisme Antimicrobial peptides are phylogenitically ancient components of innate defense mechanisms of both invertebrates and vertebrates. In the context of growing prevalence of antibiotic-resistance of bacterial strain, the AMP can be considered as potential new therapeutical candidates. The NY15 construction was based on the Leucrocin I (NGVQPKY) sequence. Leucrocin I ( (BBa_K2278022) comes from Siamese crocodile white blood cells and shows a good antibacterial activity towards Vibrio cholerae. The sequence of Leucrocin I was improved to enhance the antimicrobial properties which depends on net charge (the AMP binds to a negatively charged membrane), length, structure, hydrophobic percentage (to insert and permeabilize the microbial membrane). To do that Yamaska and al. 2013 added hydrophobic amino acids (A4, L6, F7, V8, F11) and positive change amino acids (K2, K3, K13) to Leucrocin I sequence. NY15 (NKKAGLFVVQFPKKY).

Figure 1: 3D predicted structure of NY-15 antimicrobial peptide modeled on line with Mobyle portal

The mechanism of action of the NY15 has been observed by Transmission electron microscopy. The AMPs bind and insert to bacterium membranes to create pores in it, leading to the lysis of the cells.

2- Usage in iGEM projects

The part was designed to constitutively produce the NY15 AMP with a yeast promoter. The α-factor (BBa_K1800001) sequence contains a RBS and a signal sequence to secrete the produced peptides.

Experiments

1- Molecular biology

The gene was placed in silico under the control of the p promoter (BBa_R), a strong RBS (BBa_B0034) and a terminator (BBa_B1006). IDT performed the DNA synthesis and delivered the part as gBlock.  The construct was cloned by conventional ligation into pSB1C3 plasmid and transformed into E. coli Dh5 alpha strain. X transformants were obtained.

Analysis of the restriction map

Figure 2: title Digested plasmids are electrophoresed through an 0.7% agarose gel. The desired plasmids lengths are in parentheses. pSB1C3 (2029bp the other band correspond to a xxx bp insert)

Sequencing

Figure 3: Sequencing of pSB1C3_ 1500 ng of plasmid are sequenced. X oligos were used to perform the sequencing. The obtained sequence were blast on the BBa_K2278021 sequence with the iGEM sequencing online tools.

The sequencing show a

2- Expression in vivo

sous titre

Protocole

Characterization

1- Validation of

description

manip1

Image stylée

Figure title légende

Interprétation

manip2

Image stylée

Figure plus de figure !

interprétation

Discussion :

2. 2ème approche

Figure Solid results légende de qualité

brillante analyse

Discussion :

des perspectives éclectiques

BBa_K2278021