Translational_Unit
Part:BBa_K2278021
Designed by: Paul ZANONI Group: iGEM17_INSA-UPS_France (2017-10-08)
D-NY15 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 in strain.
1- Biological background
Mécanisme 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). 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.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 (BBa_R), a strong RBS (BBa_B0034) and a terminator (BBa_B1006). IDT performed the DNA synthesis and delivered the part as gBlock. The construct was cloned by conventional ligation into pSB1C3 plasmid and transformed into E. coli Dh5 alpha strain. X transformants were obtained.
Analysis of the restriction mapSequencing
The sequencing show a2- Expression in vivo
sous titre
Protocole
Characterization
1- Validation of
descriptionmanip1
Image styléeInterprétation
manip2
Image styléeinterprétation
Discussion :
2. 2ème approche
brillante analyse
Discussion :
des perspectives éclectiques
BBa_K2278021
[edit]
Categories
Parameters
//awards/basic_part/nominee
None |