Team:NCTU Formosa/results

Magic Power of Our Pyramidal Device

　Our device combines blue light and PBAN to achieve a powerful and specific insect attraction. Firstly,we caught a female moth which had eaten PBAN,and put it into a small beaker. We covered the beaker with plastic wrap in order to keep the moth stay in beaker. Secondly, we transferred the beaker into our device and thus start our insect test in a dark chamber. We did a long-time observation to record the amount of insect entrance (into our pyramidal device) per hour. In Fig.2-0, we can really observe the magic power in attracting insects into our device!

Fig.2-0 The entering number (into our pyramidal device) per hour showed the powerful attraction of target harmful insects into our device.

Our experiment can be divided into two categories.

1. PBAN Biobricks Test: gene recombination and protein expression.

2. Insect Tests: PBAN effect test, insect behavioral test and device test.

PBAN Biobricks Test

PBAN Gene Synthesis (Full Gene Sequence Design Process)

To capture harmful insects causing lots of damage in agriculture, we first found 9 different kinds of PBAN peptide of common harmful insects all over the world from our long literature review. Next, we got the DNA sequence of these PBANs from a biotechnology web site called NCBI (EX: PBAN Spodoptera litura：http://www.ncbi.nlm.nih.gov/protein/AAK84160.1 ) Finally, we modified every codon on the DNA sequence and designed the DNA sequence for E.coli to express a certain PBAN.

DNA Modification Process：

1. Avoid the rare codon of E.coli, and choosing high frequency codons.
　　　( Frequence Table Tool：http://www.genscript.com/cgi-bin/tools/codon_freq_table )

2. Avoid choosing the same codon when modified our designed gene sequence to prevent the E.coli using up the limited nucleotides.

3. Avoid the start codon ATG existing in the front of our DNA sequence.

4. Use Rare Codon Analysis Tool ( http://www.genscript.com/cgi-bin/tools/rare_codon_analysis ) to inspect if there is any problem to express our gene for E.coli.

Take the PBAN of Spodoptera litura for example：

Fig.2-1-1 The distribution of codon usage frequency along the length of your CDS to be expressed in your target host organism. Possibility of high protein expression level is correlated to the value of CAI - a CAI of 1.0 is considered to be ideal while a CAI of >0.8 is rated as good for expression in the desired expression organism. GenScript's OptimumGeneTM codon optimization tool can typically improve your sequence to reach a CAI of higher than 0.8 thus better chance of high level protein expression.

Fig.2-1-2 The ideal percentage range of GC content is between 30% to 70%. Any peaks outside of this range will adversely affect transcriptional and translational efficiency.

Fig.2-1-3 The percentage distribution of codons in computed codon quality groups. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. Codons with values lower than 30 are likely to hamper the expression efficiency.

5. Add iGEM standard sequence in front of and at the back of our modified DNA sequence.

6. Synthesize the modified DNA sequence of PBANs in a gene synthesis company.

PCR experiment of PBAN

For checking the size of the DNA sequence received from the gene synthesis company, we recombined each PBAN gene to PSB1C3 backbone and conducted a PCR experiment for checking each size of PBAN.

Fig.2-1-4 The PCR result of the 9 different kinds of PBAN. The DNA sequence length of PBANs are around 100～150 bp, so the PCR products should appear at 415～515 bp.

Below are biobrick serial numbers of PBAN abbrevation:

BM: BBa_K1415001　　　　MB: BBa_K1415002　　　　AI: BBa_K1415003

LD: BBa_K1415004　　　　SL: BBa_K1415005　　　　HAH:BBa_K1415006

AS: BBa_K1415007　　　　SI: BBa_K1415008　　　　　AA: BBa_K1415009

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.2-1-3 showd the correct size of the PBAN, and proved that we successful ligated the PBAN DNA sequence onto an ideal backbone.

PBAN Peptide Check by SDS Protein Electrophoresis

Moreover, for verifying all the 9 kinds of PBAN can be produced 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.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　　　　　　　　　

Fig.2-1-6.2 Protein Electrophoresis of Pcons + RBS + 4 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:

AI: BBa_K1415003　　　MB: BBa_K1415002　　　HAH:BBa_K1415006　　　SI: BBa_K1415008

These SDS PAGE results in Fig.2-1-6 showed that the band at 2~4 kDa of each PBAN, while the plasmid of Pcons+RBS wasn't appeared (the PBAN peptide is an around 30 amino acids substance). This result proves that the E.coli can produce the PBAN we chosen.

Blue Light Fluorescence / Bacteria Growth Test

To predict the PBAN expression in E.coli by computer modeling, we next tested PBAN biobricks along with blue fluorescence protein. We tended to estimate 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 as the prediction of the PBAN expression in E.coli. (See more details in our Modeling Page). This is the blue fluorescence expression curve and bacterial growth curve (OD 600) below in long time, we used these data to predict our PBAN expression in E.coli.

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).

In Fig.2-1-5, we can clearly see the blue fluorescence expressed by the E.coli is different from the control without BFP expressed.

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:

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

Process to Get PBAN from E.coli

Fig2-2-1 The Process of Getting PBAN from our E.coli

In order to obtain PBAN from our E.coli, we first cultivated the E.coli that contains our constructed plasmid of Pcons + RBS + One Kind of PBAN for 12 hr. Then, we smashed the E.coli with sonicator and centrifuged the solution to allow the PBAN to be divided into the supernatant. Finally, we took the supernatant for autoclave process to avoid biosafety issues. As we all know, PBAN is a very simple and short peptide so we assumed that it will not be degraded after the autoclaved treatment. In advance, we also purified PBAN with HPLC from the supernatant ( after the autoclaved treatment ) and diluted the pure PBAN powder with 1 liter pure water for our PBAN effect test. Because PBAN can stimulate the maximum production of pheromone in very few amount ( pmol ), therefore, we don't have to worry that our PBAN concentration will be inadequate after dilution with 1 liter pure water ( actually the concentration is up to 1.5mg/L ). This is all the process of how we obtain PBAN from the E.coli.

fig.2-2-2
We put the PBAN solution for autoclaving to avoid biosafety problems.

fig.2-2-4
We dilute the PBAN powder with 1 liter of pure water.

fig.2-2-3
This is the PBAN powder which we purify with HPLC.

Insect Tests

Behavior of Target Insects After PBAN Treatment

To realize what kind of behaviors that female moth would show after releasing pheromone by eating PBAN, we put one female moth into a beaker for observation. In this beaker, it was divided into two parts by using a piece of plastic wrap. The bottom part contained the PBAN solution prepared by ourselves, and the upper part was a space for moth to stay. To let the moth suck the PBAN solution, we put some cotton soaked with PBAN solution through both parts of the beaker. After all equipments had been set, we put a 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 such as flapping their wings. In this observation, the sample moths including Spodoptera litura, Mamestra brassicae and Helicoverpa armigera Hubner were caught in Sunny Morning organic farm.

We observed that as long as the target female moth ate the PBAN, tons of PBAN can be absorbed in the moth's body in high posibility and thus, the PBAN could stimulate the moth's pheromone gland to produce pheromone and made the moth rut. As soon as the moth rutted, it would flap its wings rapidly and move its tail upward slightly.

These movies show the behaviors of 3 different kinds of female moths after eating their specific target PBANs. Each moth definitely expressed an exciting status and all flapped their wings rapidly.

Effective attraction after PBAN treatment

After observing the behaviors female moth showed, we conducted this experiment in the moth box(show in Fig.2-3-1) to check the attractive effect of our idea. We hoped that the female moth can not only become excited, flap its wings but also actually attract male moths to aggregate together after eating the PBAN. We used two beakers which are the same as what we used in the former experiment. One contained PBAN solution and the other contained only sucrose solution as control, we put one beaker at one edge of the moth box and the other one at the opposite edge. This time, we did a long time observation and took a picture with our camera. In Fig.2-3-1, the female moth ate the PBAN then attracted more male moths than the one without eating our PBAN. Thus, Fig.2-3-1 can prove the fact that the female moth ate our PBAN then release much sex pheromone to attract many male moths. In addition, we also conducted a simple test to compare the luring effect of female moths eating PBAN solution with the luring effect of female moths eating sucrose solution (the moth favorite food). Also, we can see the conspicuous effect again.

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.

Spodoptera Litura Hobby for Temperature and Light

　Light can be used to attract many kinds of harmful insects.

Temperature is the environmental factor which the farmer can not change practically. We want to use the computer modeling to deeply explore the relationship among light, temperature and the moths' hobby. In the future, we hope that farmers can choose the appropriate light according to temperature condition and even the kind of moths when using our device. For this, we chose the average temperature range in Taiwan in a year, and most common harmful insect, Spodoptera Litura to conduct this test (Fig.2-3-3 below), which we wanted to use to model the relationship among light, temperature and the moths' hobby with ANFIS (See detail in the device modeling page).

Fig.2-3-3 Result of 30 numbers of Spodoptera Litura's Hobby testing for temperature and light (there are some moths staying in the bottom every testing). In this table, we can see blue light really have a steady attraction to Spodoptera Litura in different temperature condition.

Fig.2-3-4 Modeling tool (CCW No.1) for this testing. We use blue, green, red, yellow,Positive Control: white at the same time in different temperature conditions for our device modeling testing. Spodoptera Lituras were packed into the central barrel. Every testing was followed by continuous beating on barrel for 5 min in order to make the moths fly.

　Fig.2-3-3 shows blue light have steady attraction to our target harmful moths, Spodoptera Litura, in any temperature condition. Thus, we decided to use blue LED light in our device design.