Part:BBa_K1415003

PBAN (Agrotis ipsilon)

Introduction: PBAN (Pheromone Biosynthesis Activating Neuropeptide)

Fig.1-1 A coding gene of a Agrotis ipsilon'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.coil, 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 Agrotis ipsilon's PBAN.

See our expanding PBAN(Agrotis ipsilonr) parts collection: Pcons+B0034+PBAN(Agrotis ipsilon) and Pcons+B0034+PBAN(Agrotis ipsilon)+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: Cotton bollworm (Agrotis ipsilon)

Fig.2-1 Introduction of Agrotis ipsilonr

The experiment of PBAN

Fig.2-2 The PCR result of the PBAN-AI. The DNA sequence length of PBANs are around 100～150 bp, so the PCR products should appear at 415～515 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 part-PBAN(Agrotis ipsilon)

Application of the part

Fig.3-1 P_cons+RBS+PBAN(Agrotis ipsilon)

To verify that the PBAN of Agrotis ipsilon 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 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

　

Fig.4-1 Biobrick of Pcons + RBS + PBAN(Agrotis ipsilon) +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 Pcons + 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. Agrotis ipsilon') + RBS +BFP +Ter]]

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

Fig.4-4 The growth curves of PBAN(MB)

modeling

Fig.4-5 Modeling result of P_cons + RBS + PBAN(Agrotis ipsilon) + 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(Agrotis ipsilon) + 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.4-6 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.