Difference between revisions of "Part:BBa K2278023"
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<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/5/5d/COT2inhibition_.png" width = "500"/><figcaption> Figure 5: <b>AMP halo assay</b> 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.</figcaption></figure> | <figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/5/5d/COT2inhibition_.png" width = "500"/><figcaption> Figure 5: <b>AMP halo assay</b> 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.</figcaption></figure> | ||
− | 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. | + | 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. |
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Revision as of 11:57, 17 October 2017
Lecrocin I antimicrobial peptide with Alpha-Factor Secretion Signal
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 244
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.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 mapSequencing
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.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