"
Team:NCTU Formosa/results
From 2014.igem.org
Contents |
Magic Power of Our Pyramidal Device
Our experiment can roughly divided into two categories.
1. About E.coli. Aspects：gene recombination and protein expression.
2. About Insect Aspects：PBAN effect testing、insects' habbit testing and our device testing
E.coli. Aspects
DNA Synthsis of 9 different kinds of PBAN
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 our 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：
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.
PCR for 9 different kinds of PBAN
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 ro check each PBAN size.
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.
SDS Protein Electrophoresis of 9 different kinds of PBAN
In advance, for proving all the 9 different kinds of PBAN can be produced by our E.coli., we smashed the E.coli.,containg our PBAN with Sonicator and take the supernatant divided from the bacterial pellet after centrifuged to do 20% gel SDS protein electrophoresis.
Because PBAN peptide is an around 30 amino acids substance, in fig.2-1-5 we can see the band at 2~4kDa, and the E.coli. containg Pcons+RBS Plasmid don't. This result proves that our E.coli. can really produce our PBAN.
BFP Fluorescence / OD 600 Bacteria Growth Testing
We wanted to use computer modeling to help us predicting PBAN expression in E.coli., so we decided to use BFP flourescence to reach our goal. Our thought is that we can compute the average value of the BFP 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 BFP 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..
Insect Aspects
Insect experiment protocol
In order to test our PBAN device, we had to conduct many insect experiments, we chose Spodoptera Litura as experimental lead in modeling experiments and PBAN tests because the vitality of Spodoptera Litura in Taiwan just peaked at that time. Some things needed to be prepared well before every test and experiment, what we needed are many devices to keep and catch Spodoptera Litura and fed them, the devices we used was listed below:
-
1.Several insect-catching nets as well as insect-catching boxes
2.An giant experimental box made by acrylic boards as the space for PBAN experiments and storing Spodoptera Litura , (長寬高)
3.The "food" provided for Spodoptera Litura, including some flowers which contained nectar and 10%~15% sucrose solution
4.A barrel equipped with several lights, we used this barrel in experiment of Spodoptera Litura hobby corresponding to changes of temperature and light
5.Spodoptera Litura, we caught many Spodoptera Litura from organic farm by light traps and insect-catching nets. In addition, we also bred them by ourselves, in a word, we had enough amount of Spodoptera Litura to conduct experiment
6.PBAN solution (濃度)
7.A professional digital camera, we used it to take many photos and videos, sometimes we also used smartphone to do that.
8.Last but not least, our device.
Behavior of Target Insects Eating Our PBAN
To realize what kind of behavior female moth would show after eating PBAN and realesing pheromone, we put one or several female moths into a beaker which was divided into layers and the lower layer contained PBAN solution made by ourselves, and then we sealed the beaker with handi-wrap, the toilet paper which was soaked with PBAN solution connected the both layers of the beaker helped female moth suck PBAN solution, we started filming as soon as we observed that the female moth show obvious behavior, the moth sample included Spodoptera litura、 Mamestra brassicae、 Helicoverpa armigera Hubner which we caught in Sunny Morning organic farm. We think as long as the target female moth eats our PBAN, tons of PBAN can be absorbed in the moth's body in high posibility and thus, our PBAN can stimulate the moth's pheromone gland to produce pheromone and make our moth ruts. As soon as the moth is in rut, it will flap its wings rapidly and move its tail upward slightly. Thus, before the PBAN effect testing, we decided to observe the behavior of our target female moth eating our PBAN and we hoped to see the expected behavior result.
<p> These movie showed after two different kinds of female moths eats our PBAN SL & PBAN MB, these two moths really became excited and all flapped their wings rapidly.</p>
Effect Testing of Our PBAN
After observing the behavior female moth showed, we decided to conduct this experiment in the moth box to check the attractive effect of our idea, we hope our female moth can not only become excited, flapping its wings but also actually attract many male moths to aggregate together after eating our PBAN. we used two beakers which are the same as that we used in the former experiment, one contained PBAN solution and the other contained only sucrose solution as control and was put at two sides of the moth box. This time, we did a long time observation and recorded with our video camera below.
Fig.2-2-1 shows the female moth eating our PBAN could attract more male moths than the female moth without eating our PBAN did. Thus, fig.2-2-1 can prove the fact that the female moth eating our PBAN can release much sex pheromone, and attract many male moths.
Testing of 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 2-2-2 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 2-2-2 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.
