Team:NCTU Formosa/results

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Results

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For our project (aim for capturing harmful insects) this year, we first found 9 different kinds of PBAN peptide of common agricultural harmful insects in the world from many reference papers. Then, we used these peptides to surf the NCBI and found the DNA sequence from the certain insect. ( 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. Don't choose the same codon to modify our designed gene many times, or our E.coli will not have enough nucleotides to replicate it.

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 PBAN 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. Let the gene synthesis company synthesize our modified DNA sequence.

PBAN PCR Result

For checking the size of the DNA sequence getted from the gene synthesis company, we recombined each PBAN gene to PSB1C3 backbone and do PCR to check each PBAN size.

Fig.2-1-4 The PCR result of our 9 different kinds of PBAN. Because our PBAN DNA sequence length is around 100～150 bp. The PCR result should be 415～515 bp.

Because our PBAN DNA sequence length is around 100～150 bp. The PCR result should be 415～515 bp. The Fig.2-1-3 shows the correct size of our PBAN, and proves we succeed in ligating our PBAN gene to the ideal backbone.

PBAN Peptide SDS Protein Electrophoresis

In advance, for proving all the 9 different kinds of PBAN can be produced by our E.coli, we smashed the E.coli, containing our PBAN with Sonicator and take the supernatant divided from the bacterial pellet after centrifuged to do 20% gel SDS protein electrophoresis.

Fig.2-1-6.1 Protein Electrophoresis of Pcons + RBS + 5 different kinds of PBAN (Control: E.coli containg Pcons+RBS Plasmid) Because each PBAN is an around 30 amino acids peptide, we can see the band at 2~4 kDa

Fig.2-1-6.2 Protein Electrophoresis of Pcons + RBS + 4 different kinds of PBAN (Control: E.coli containg Pcons+RBS Plasmid) Because each PBAN is an around 30 amino acids peptide, we can see the band at 2~4 kDa

Because PBAN peptide is an around 30 amino acids substance, in Fig.2-1-6 we can see the band at 2~4 kDa, and the E.coli containg Pcons+RBS Plasmid don't. This result proves that our E.coli can really produce our PBAN.

Blue Light Fluorescence / Bacteria Growth Test

We wanted to use computer modeling to help us predicting PBAN expression in E.coli, so we decided to use blue light flourescence to reach our goal. Our thought is that we can compute the average value of the blue light flourescence expression value of the biobrick part (above) and of the Pcons + RBS + BFP + Ter and regard the average value as the prediction of our PBAN expression in E.coli (Pcons + RBS + PBAN + Ter) (More detail can be seen in our Modeling Page). This is the blue light flourescence expression curve and bacterial growth curve (OD 600) below in long time, we use these data to predict our PBAN expression in E.coli.

Fig.2-1-5 Blue light Flourescence of Pcons + RBS + 9 different kinds of PBAN (control: E.coli containg Pcons+RBS Plasmid).

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).

Process to Get PBAN from Our E.coli

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

　　For getting PBAN from our E.coli, we first cultivated the E.coli containing a plasmid composited of Pcons + RBS + One Kind of PBAN for 12 hr. Then, we smashed the E.coli with sonicator and centrifuged the solution to let the PBAN divided into the supernatant. Finally, we take the supernatant diluted by 1 liter pure water for autoclaving to avoid biosafety problems. (Because PBAN is a very simple and short peptide, we suppose that it will not be degraded after the autoclave treatment). This is all the process that we get PBAN from the E.coli.

Fig2-2-2 We put the PBAN solution for autoclaving to avoid biosafety problems.

Fig2-2-3 We succeed in getting PBAN from E.coli after autoclaving treatment.

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 or several female moths into a beaker which was divided into two layers. The lower layer contained the PBAN solution made by ourselves. Then we sealed the beaker with handi-wrap, the toilet paper which was soaked with PBAN solution connected the both layers of the beaker helping female moth to suck the PBAN solution. At the time, we started filming as soon as we observed that the female moth show obvious behaviors such as flapping their wings. In this observation, the sample of moth 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 movie show the behavior of two different kinds of female moths after eating the PBAN SL and PBAN MB. These two moths definitely expressed an exciting status and all flapped their wings rapidly.

Powerful Effect of PBAN

After observing the behaviors female moth showed, we conducted this experiment in the moth box to check the attractive effect of our idea. We hope 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 be put at two sides of the moth box. 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 releasing much sex pheromone to attract many male moths. In addition, we also conducted a simple test to compare our female moths eaing our PBAN with the sucrose solution (the moth favorite food) luring factor. Also, we can see the gigantic PBAN 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 our PBAN can release much sex pheromone, and attract many male moths.

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

Spodoptera Litura Hobby for Temperature and Light

　Light can be probable of attracting target harmful female 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 choose the average temperature range in Taiwan in a year, and most common harmful insects, Spodoptera Litura to conduct this test (Fig.2-3-3 below), which we want 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 into our device design.