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The sequencing successfully validated the sequence of the biobrick.  
 
The sequencing successfully validated the sequence of the biobrick.  
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=='''Characterization'''==
 
=='''Characterization'''==
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<h3 id="RT"> Toxicity assay  </h3>
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<h3 id="RT">2. Toxicity assay  </h3>
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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
 
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
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The toxicity assay did not reveal any activity of the cOT2 AMP
 
The toxicity assay did not reveal any activity of the cOT2 AMP

Revision as of 07:00, 16 October 2017


Lecrocin I 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 coT2 AMP with Alpha-Factor Secretion Signal in a yeast organism 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. Crocodile ovotransferrin 2 peptide (cOT2) is an engineered peptides coming from the Siamese crocodile. It bears the natural sequence of cot1 and has been extended based on the C. siamensis transferrin amino sequence to increase its natural antimicrobial activity The peptide is a 29 amino acid residue : KKSCHTGLKKSAGWVIPIGTLVKNGIIVR The mechanism of action of the cot2 has been observed scanning electron microscopy. This cationic and amphipathic molecules is able to attach to and insert into membrane bilayers to form pores.

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

2- Usage in iGEM projects

The part was designed to constitutively produce the cOT2 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.

Characterization

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 The toxicity assay did not reveal any activity of the cOT2 AMP Source