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

Mechanisme 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: (A) 3D predicted structure of NY-15 antimicrobial peptide modeled on line with Mobyle portal. (B) Example of an AMP model of intracellular killing mechanism : the barrel-stave model of antimicrobial-peptide-induced killing (Brogden, 2005)

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.

Figure 1: Scanning electron micrographs of Vibrio cholerae treated with peptides (a) control control bacteria c) bacteria treated with NY15 AMP (Yaraksa and al., 2014)

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 IDT performed the DNA synthesis and delivered the part as gBlock.  The construct was cloned by conventional ligation into pSB1C3 plasmid The construction was then inserted on plasmid pPICZa and integrated in the yeast genome.

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 successfully validated the sequence of the biobrick.

2- Expression in vivo

sous titre

Protocole

Characterization

1- Validation of D-NY15 expression in Pichia pastoris by RTq-PCR

description

RTqPCR

. (IMAGE)

Figure RTq-PCR of D-NY15 The amount of fluorescence provided by the RTq-PCR with the D-NY15 amorces is raising after few cycles (8.32 +/- 0.03) whereas the negative control (Ppic only) start to be amplified at 29 cycles (non specific amplification). This mean that mRNA of DNY15 have been produced.

2. Toxicity assay

The engineered yeast were used in a halo assay against V. harveyi as the target of AMPs. A paper soacked with a yeast solution was placed on the plate and V. harveyi growth in the viscinity of the yeast patch was followed

Figure AMP halo assay Positive control is performed with chloramphenicol (25 g/L), the negative control is performed with empty plasmid integrated in P. pastoris, the assay is performed with plasmid containing BBa_K2278021 integrated in P. pastoris/ The yeast solution was concentrated 10 times

Conclusion :

Here, we show both the capacity of Pichia pastoris to produce antimicrobial peptides and the efficiency of the crocodile peptide D-NY15 to inhibit Vibrio harveyi growth. cOT2 and Leucrocine did not appear as inihibitory in the same conditions. Still, we successfully produced an antimicrobial peptide that is active.

Perspectives :

: To go further, inhibition test should be now performed in liquid assay to be closer to the conditions in which we want to use these strains.