Part:BBa_K2278021

D-NY15 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 an antimicrobial peptide from the crocodile Crocodylus siamensis.

1- Biological background

Antimicrobial peptides (AMP) are phylogenetically ancient components of the innate defense of both invertebrates and vertebrates. In the context of growing bacterial antibiotic-resistance, these AMP are considered as potential new therapeutical candidates.
The D-NY15 construction was based on the small peptide Leucrocin I sequence (NGVQPKY ; Yaraksa et al., 2013). 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, and hydrophobic percentage (to insert and permeabilize the microbial membrane). To do that Yaraksa et al added hydrophobic amino acids (A4, L6, F7, V8, F11) and positively charged amino acids (K2, K3, K13) to Leucrocin I sequence. This results in D-NY15 (NKKAGLFVVQFPKKY ; figure 1).

Figure 1: **(A) 3D predicted structure of NY-15 antimicrobial peptide** modeled on line with Mobyle portal. **(B) Example of an AMP model of intracellular killing mechanism** : the barrel-stave model of antimicrobial-peptide-induced killing (Brogden, 2005)

The mechanism of action of D-NY15 has been observed by transmission electron microscopy. The AMP binds and inserts to bacterium membranes to create pores, leading to the lysis of the cells (figure 2).

Figure 1: **Scanning electron micrographs of Vibrio cholerae treated with peptides** (a) control bacteria c) bacteria treated with D-NY15 AMP (Yaraksa *et al*., 2014)

2- Usage in iGEM projects

The part was designed to constitutively produce the D-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 an alpha factor signal. 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

Sequencing

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_K2278021 in pichia pastoris** To verify the function of the new Biobrick, we performed a colony PCR using primers to anneal the construction. To check the length of the resulting PCR products, we electrophoresed the reactions through an 0,7% agarose gel. Lane 1 correspond to 1kb DNA ladder (new England bolas, Inc)

Expression of D-NY15 AMP

D-NY15 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

1- Validation of D-NY15 expression in Pichia pastoris by RTq-PCR

Figure **RTq-PCR of D-NY15** The amount of fluorescence provided by the RTq-PCR with the D-NY15 amorces is raising after few cycles (8.32 +/- 0.03) whereas the negative control (Ppic only) start to be amplified at 29 cycles (non specific amplification). This mean that mRNA of DNY15 have been produced.

2. 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_K2278021 integrated in P. pastoris/ The yeast solution was concentrated 10 times

Conclusion :

Here, we show both the capacity of Pichia pastoris to produce antimicrobial peptides and the efficiency of the crocodile peptide D-NY15 to inhibit Vibrio harveyi growth. We have successfully produced an antimicrobial peptide that is active.

Perspectives:

To go further, inhibition test should be now performed in liquid assay to be closer to the conditions in which we want to use these strains.

Design Notes

A pGAP promoter is present on the pSB1C3 vector before the construction. It makes genome recombination easier in Pichia pastoris genome.

Part:BBa_K1800001: Alpha-Factor Secretion Signal

Source

The peptides DNA sequence has been obtained by reverse translate the amino acid sequence of the NY15 proposed by Yaraksa et al., 2013 which was determined by mass spectrometry analysis.

References

Yaraksa, N., Anunthawan, T., Theansungnoen, T., Daduang, S., Araki, T., Dhiravisit, A. and Thammasirirak, S. (2013). Design and synthesis of cationic antibacterial peptide based on Leucrocin I sequence, antibacterial peptide from crocodile (Crocodylus siamensis) white blood cell extracts. The Journal of Antibiotics, 67(3), pp.205-212.

Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria. Nat Rev Microbiol. 3:238-50.