Part:BBa_K2278022

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 in strain.

1- Biological background

Antimicrobial peptides are phygenitically 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. The part was designed to constitutively produce the leucrocin I AMP with a yeast promoter. The α-factor sequence contains a RBS and a signal sequence to secrete the produced peptides. The pGAP promoter is used because it makes genome recombination easier in Pichia pastoris genome. The restriction enzyme sites are set up to extract individually each components of the plasmid. It belongs to the respond module in the Croc’n cholera project of iGEM INSA-UPS-France 2017 Mécanisme

Figure 1: figure caption

2- Usage in iGEM projects

The BBa_K2278022 cames from the module of the Croc’n cholera project (team INSA-UPS-France 2017) It was designed to produce

The part includes

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 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_K2278022 sequence with the iGEM sequencing online tools.

The sequencing show a

2- Expression in vivo

sous titre

Protocole

Characterization

1- Validation of

description

manip1

Image stylée

Figure title légende

Interprétation

manip2

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Figure plus de figure !

interprétation

Discussion :

2. 2ème approche

Figure Solid results légende de qualité

brillante analyse

Discussion :

des perspectives éclectiques

BBa_K2278022