Part:BBa_K2278023

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(GAP) 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: Validation of BBa_K2278023 in pSB1C3 restriction map Digested plasmids are electrophoresed through an 0.7% agarose gel. The desired plasmids lengths are in parentheses. pSB1C3 (2029bp) the other band correspond to the 300 bp insert.

Sequencing

Figure 3: Sequencing of BBa_K2278023 in pSB1C3 1500 ng of plasmid are sequenced. 2 oligos were used to perform the sequencing. The obtained sequence were blast on the BBa_K2278023 sequence with the iGEM sequencing online tools.

The sequencing successfully validated the sequence of the biobrick. The sequencing successfully validated the sequence of the biobrick.

2- Expression in vivo

Integration in Pichia pastori genome

Protocole

The biobrick was placed in silico under the control of p(GAP) promoter (BBa_K431009) and was cloned in pPICZalpha vector, a good expression vector for Pichia pastoris. The plasmid was then linearized and transferred in Pichia pastoris by electroporation. The integration is predicted to be at the p(GAP) location. Indeed, the p(GAP) promoter makes genome recombination easier in Pichia pastoris genome thanks to its homology site.

Figure 3: Integration of p(GAP)+BBa_K2278023 in pichia pastoris To verify the function of the new Biobrick, we performed a DNA extraction. To check the length of the resulting DNA, we digested the DNA with EcoRI and Ncos restriction enzyme and electrophoresed the reactions through an 0,7% agarose gel. Lane 1 correspond to 1kb DNA ladder (new England bolas, Inc)

Expression of cOT2 AMP

cOT2 production was performed with the P. pastoris YPD 40 g/L glucose and grown for 4 days at 30 °C in an agitating incubator. 15mL of each supernatant culture were stored at 4°C while 35mL were freeze-dried and then resuspended in 3.5mL of water.

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.

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_K2278023 integrated in P. pastoris.

No inhibition halo was observed around the yeast patch. The cOT2 cytoxicity can not be demonstrated. The toxicity assay did not reveal any activity of the cOT2 AMP. Source