PBAN (Spodoptera litura)

Introduction:PBAN (Pheromone Biosynthesis Activating Neuropeptide)

Fig.1-1 A coding gene of a Spodoptera litura's PBAN

Mechanism of PBAN

PBAN (Pheromone Biosynthesis Activating Neuropeptide) is one kind of peptides that can activate biosynthesis of pheromones of insects we target. Once a PBAN binds with the G-protein coupled receptor on an insect’s pheromone gland, the signal send by the G-protein coupled receptor activates the kinase and phosphatase, and then kinase and phosphatase can activate enzymes that participate in the biosynthesis of insect pheromone, which will be emitted.

Features of PBAN

1. Species-Specific: PBAN is species-specific just like pheromones, meaning that every kind of insect produces specific PBAN that only binds with it's targeted receptor, resulting in the production of a particular pheromone.
2. Small Simple Peptide: The coding sequence for a PBAN is only around 100 base pairs. To E.coli 100 base pairs is totally within its working capacity. And therefore, E.coli can be our low-cost PBAN factory. By synthesizing the DNA sequences for different PBAN into our factory, we can even produce a variety of PBANs. In addition, this factory is totally environmental friendly, unlike any pesticide we have seen.　
3. Insects' own secretion: Because PBAN is a insect's own secretion, insects could not form resistance it. In addition, it can easily trigger pheromone production by coming in contact with its receptor.

This part is a coding gene of a Spodoptera litura's PBAN.
See our expanding PBAN(SL) parts collection: Pcons+B0034+PBAN(Spodoptera litura) and Pcons+B0034+PBAN(Spodoptera litura)+B0034+BFP+J61048

Fig.1-2 Working mechanism of PBAN

Target insect:Oriental Leafworm Moth (Spodoptera litura)

Fig.1-3 Introduce of Spodoptera litura's

The experiment of PBAN

After receiving the DNA sequences from the gene synthesis company, we recombined each PBAN gene to PSB1C3 backbones and conducted a PCR experiment to check the size of each of the PBANs. The DNA sequence length of the PBAN are around 100～150 bp. In this PCR experiment, the PBAN products size should be near at 415～515 bp. The Fig.1-4 showed the correct size of the PBAN, and proved that we successful ligated the PBAN DNA sequence onto an ideal backbone.

Fig.1-4 The PCR result of the PBAN-SL. The DNA sequence length of PBANs are around 100～150 bp, so the PCR products should appear at 415～515 bp.

Application of the part

Fig.2-1-6-1 Protein Electrophoresis of Pcons + RBS + 5 different kinds of PBAN (control: plasmid of Pcons+RBS) Each peptide of PBAN is an around 30 amino acids, so we can see the band of PBANs at 2~4 kDa.

Below are biobrick serial numbers of PBAN abbrevation:

BM: BBa_K1415001　　　AA: BBa_K1415009　　　LD: BBa_K1415104

AS: BBa_K1415007　　　SL: BBa_K1415005

Behavior of Target Insects After PBAN Treatment

To investigate what behavior the female moth would show after ingesting PBAN, we put one female moth into a beaker for observation. The beaker is divided into two parts by plastic wrap. The bottom part contains the PBAN solution we prepared, and the upper part is the space for the moth to stay. We soaked cotton that spans the entire length of the beaker with the PBAN solution and sprinkle it with sugar. This way, the moth can suck on the PBAN without drowning in PBAN solution. After all the equipment is set, we put the female moth into the upper part of the beaker. At the time, we started filming as soon as we observed the female moth showing obvious behaviors of sexual stimulation such as flapping their wings. In this observation, the sample moth is Spodoptera litura which we caught in Sunny Morning organic farm.

We observed that the moth could absorb the PBAN in the solution through ingestion, and that the PBAN could stimulate the moth's pheromone gland to produce pheromone. As soon as the moth is sexually excited, it would flap its wings rapidly and move its tail slightly upward .

These movies show the behaviors of female moth after ingesting its separate PBANs. The moths clearly became excited and flapped their wings rapidly.

Fig.2-3-1 Negative Control: Female moth without eating PBAN (Number = 0). Experiment: Female moth eating our PBAN (Number = 11). In this picture, we can see the PBAN effect that the female moth eating PBAN solution can release much sex pheromone, and attract many male moths.

Fig.2-3-2 Negative Control：sucrose solution, Experiment：Female moth eating PBAN solution. Also, we can see the PBAN effect again from this picture.

Fig.3-4-1 Biobrick of Pcons + RBS + PBAN(SL) + BFP + Term.

Fig.2-1-7 The growth curve of E.coli containing Pcons + RBS + 9 different kinds of PBAN + RBS + BFP + Ter plasmid (control is the competent cells which can not emit blue light).

Fig.2-1-8 The blue light fluorescence expression curve of E.coli containing Pcons + RBS + 9 different kinds of PBAN + RBS + BFP + Ter plasmid (control is the competent cells which can not emit blue light).

Fig.2-1-5 Blue Fluorescence of Pcons + RBS + 9 different kinds of PBAN (control: E.coli containg Pcons+RBS Plasmid).　Below are biobrick serial numbers of PBAN abbrevation:</p> SL: BBa_K1415005　　　　BM: BBa_K1415001　　　　MB: BBa_K1415002

AI: BBa_K1415003　　　　LD: BBa_K1415004　　　　HAH:BBa_K1415006

AS: BBa_K1415007　　　　SI: BBa_K1415008　　　　AA: BBa_K1415009

modeling

Fig.3-4-2 Modeling result of Pcons + RBS + PBAN(SL) + BFP + Ter. The blue line is the expression profile of the theoretical biobrick. And the green line is the expression data of Pcons + RBS + PBAN(SL) + BFP + Ter. And the red line is the adjusting line from the green and blue one. This line represent the correcting line of theoretical data and real condition data which can make our model not only fit the theoretical condition but also stay away from experimental bias.

The device we design and working mechanism