Part:BBa_K1415005

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 its specific receptor, resulting in the production of a particular pheromone.

2. Small and simple: The coding sequence for a PBAN is only around 100 base pairs. For E.coli, 100 base pairs is totally within its working capacity. Therefore, E.coli can be a low-cost PBAN factory. By transforming the DNA sequences for different PBAN into the E.coli, we can even gain a variety of PBANs. 　

3. Secreted directly: Because PBAN is secreted by the insect itself, the insect would not form a resistance to it compare to use pesticide.

Together, using PBAN is totally a environmental friendly way for solving harmful insects problems with easily triggering pheromone production by contacting with its receptor.

This part is a coding gene of Spodoptera litura's PBAN.

See our expanding PBAN(Spodoptera litura) parts collection: P_cons+B0034+PBAN(Spodoptera litura) and P_cons+B0034+PBAN(Spodoptera litura)+B0034+BFP+J61048

Fig.1-2 Working mechanism of PBAN

Reference:

Ada Rafaeli, Pheromone biosynthesis activating neuropeptide (PBAN): Regulatory role and mode of action, General and Comparative Endocrinology 162 (2009) 69–78.

Target insect: Oriental Leafworm Moth (Spodoptera litura)

Fig.2-1 Introduction of Spodoptera litura

The experiment of PBAN

Fig.2-2 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 300～350 bp.

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.2-2 showed the correct size of the PBAN, and proved that we successful ligated the PBAN DNA sequence onto an ideal backbone.

Fig.2-3 The plate of our PBAN(Spodoptera litura)

Application of the part

P_cons+RBS+PBAN(Spodoptera litura)

Fig.3-1 P_cons+RBS+PBAN(Spodoptera litura)

To verify the PBAN of Spodoptera litura can be expressed by the E.coli, we conducted a SDS protein electrophoresis experiment. We first smashed the E.coli containing the PBAN with a sonicator and then took the supernatant divided from the bacterial pellet by centrifugation. Finally, we used the supernatant to run a SDS protein electrophoresis in a 20 % SDS gel.

Fig.3-2 Protein Electrophoresis of P_cons + RBS + 5 different kinds of PBAN (control: plasmid of P_cons+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 .

This video shows the behaviors of female moth after ingesting its separate PBANs. The moths clearly became excited and flapped their wings rapidly.

Fig.3-3 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.3-4 Negative Control:sucrose solution, Experiment:Female moth eating PBAN solution. Also, we can see the PBAN effect again from this picture.

Fig.4-1 Biobrick of P_cons + RBS + PBAN(Spodoptera litura ) + RBS + BFP + Ter.

To predict the PBAN expression in E.coli by computer modeling, we next tested PBAN BFP biobricks. We obtained the average expressive value of the blue fluorescence in the biobrick part (above) and also the control part of P_cons + RBS + BFP + Ter. Therefore, we can use the average value to generate predictions of the PBAN expression in E.coli. Below is the blue fluorescence expression curve and bacterial growth curve (OD 600) in a long period of time. We used these data to predict the PBAN expression in E.coli.

Fig.4-2 Blue Fluorescence of P_cons + RBS + PBAN(Spodoptera litura) +RBS + BFP + Ter.

Fig.4-3-1 The blue light fluorescence expression curve of E.coli containing Pcons + RBS + PBAN(SL) + RBS + BFP + Ter plasmid (control: competent cells that cannot emit blue light).

Fig.4-3-2 The growth curves of PBAN(SL)

Modeling

Fig.4-5 Modeling result of P_cons + RBS + PBAN(Spodoptera litura) + BFP + Ter. The blue line is the expression profile of the theoretical biobrick. And the green line is the expression data of P_cons + RBS + PBAN(Spodoptera litura) + 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

Fig.5-1 Our Project Overview.

In our project, we will biologically synthesize PBAN with our E.coli. We store the PBAN inside a trapping device. In the device, there will be appropriate lighting and nutrient sources that will attract insects.

Once an insect is attracted into our device and ingests the nutrient sources we provide, it will also inevitably come in contact with our PBAN. As the PBAN works and activates the pheromone synthesis of the attracted insect, more of this species of insect’s counterparts will be attracted and later captured.

Owing to the first feature mentioned above, PBAN are species-specific, which means that it doesn't matter if other kind of insect fly into our device and eat PBAN, because the insects we don't want to catch will not be stimulated by PBANs to produce pheromone; our PBAN are only for what we want to catch, and we are sure that our method won't affect other kinds of insects.