Difference between revisions of "Part:BBa K2278022"
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=='''Introduction'''== | =='''Introduction'''== | ||
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| + | This DNA biobrick was designed in order to produce Lecrocin I AMP in a yeast organism <i></i> strain. | ||
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| + | <h3 id="RT"> 1- Biological background </h3> | ||
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| + | 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. | ||
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Leucrocin I from Siamese crocodile white blood cells shows a good antibacterial activity towards Vibrio cholerae. | Leucrocin I from Siamese crocodile white blood cells shows a good antibacterial activity towards Vibrio cholerae. | ||
The peptide is a 7 amino acid residue : NGVQPKY with a molecular mass around 806.99 Da. | The peptide is a 7 amino acid residue : NGVQPKY with a molecular mass around 806.99 Da. | ||
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| − | + | The mechanism of action of the Leucrocin I has been observed with fluorescence and electron microscopy This cationic molecules and can target bacterium membranes, to create pores in it, leading to the lysis of the cells. | |
| − | <figure><p style="text-align:center;"> <img src =" | + | <figure><p style="text-align:center;"> <img src ="https://static.igem.org/mediawiki/parts/4/4d/Yaraksa14.png" width = "600" /> <figcaption> Figure 1: <b>Scanning electron micrographs of Vibrio cholerae treated with peptides </b> (a) control control bacteria c) bacteria treated with NY15 AMP (Yaraksa and al., 2014)</figcaption> </figure> |
<h3 id="RT"> 2- Usage in iGEM projects </h3> | <h3 id="RT"> 2- Usage in iGEM projects </h3> | ||
| − | + | The part was designed to constitutively produce the leucrocin I AMP with a yeast promoter. The α-factor (BBa_K1800001) sequence contains a RBS and a signal sequence to secrete the produced peptides. | |
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=='''Experiments'''== | =='''Experiments'''== | ||
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<h3 id="RT"> 1- Molecular biology </h3> | <h3 id="RT"> 1- Molecular biology </h3> | ||
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| − | The gene was placed in silico under the control of the p promoter | + | 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 and | + | |
| + | The construct was cloned by conventional ligation into pSB1C3 plasmid | ||
| + | The construction was then inserted on plasmid pPICZa and integrated in the yeast genome. | ||
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<p><b>Sequencing </p></b> | <p><b>Sequencing </p></b> | ||
| − | <figure><p style="text-align:center;"><img src="" width = "500"/><figcaption> Figure 3: <b>Sequencing of pSB1C3_ </b> 1500 ng of plasmid are sequenced. X oligos were used to perform the sequencing. The obtained sequence were blast on the | + | <figure><p style="text-align:center;"><img src="" width = "500"/><figcaption> Figure 3: <b>Sequencing of pSB1C3_ </b> 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. </figcaption></figure> |
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| − | + | The sequencing successfully validated the sequence of the biobrick. | |
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Revision as of 06:54, 16 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 Lecrocin I AMP 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. Leucrocin I from Siamese crocodile white blood cells shows a good antibacterial activity towards Vibrio cholerae. The peptide is a 7 amino acid residue : NGVQPKY with a molecular mass around 806.99 Da. The mechanism of action of the Leucrocin I has been observed with fluorescence and electron microscopy This cationic molecules and can target 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 leucrocin I 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 mapSequencing