Difference between revisions of "Team:Wageningen UR/Results"
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<img src="https://static.igem.org/mediawiki/2016/5/51/T--Wageningen_UR--specifitytitle.jpg"></a> | <img src="https://static.igem.org/mediawiki/2016/5/51/T--Wageningen_UR--specifitytitle.jpg"></a> | ||
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| + | <p>In order to improve on existing methods, BeeT should effect <i>Varroa</i> mites only. To accomplish this we decided to make use of Cry toxins. A functional Cry toxin is only effective when specific binding occurs to the gut membrane of the target organism. Hereafter, the Cry toxins will form pores into the cell membrane, which results in cell death. As cell death occurs, the gut membrane becomes porous. Consequently, the organism dies. <sup><a href="#re1" id="refre1">1</a></sup> To find a Cry toxin active against <i>V. destructor</i> we engineered our own toxins and we searched in nature for one as well.<br><br> | ||
| + | Due to the parasitic nature of <i>Varroa</i> mites, testing Cry toxins proved to be very problematic. To overcome this problem we developed an <i>in vitro</i> test for Cry toxins. Out of the membranes of the target organism, brush border membrane vesicles (BBMVs) were made and incorporated with 6-carboxyfluorescein. A functional Cry toxins will create pores into the BBMVs, which then results in the leaking of fluorophores out of the BBMVs. Due to self-quenching behaviour of 6-carboxyfluorescein, this can be measured as an increase in fluorescence. As a proof of principle, BBMVs from the gut of <i>Tenebrio molitor</i> were made and loaded with 6-carboxyfluorescein to test the pore formation ability of Cry3Aa, which is known to be toxic to <i>T. molitor</i> larvae <sup><a href="#jz3" id="refjz3">3</a></sup>. | ||
| + | Figure 1a shows how the fluorescence increases of BBMVs incorporated with fluorophores in the presence and absence of Cry3Aa. A kinetic value could be coupled to this process. These values for multiple measurements for BBMVs in the presence and absence of Cry3Aa are shown in Figure 1b. From this can be concluded that the presence of a functional Cry protein results can be measured.</p> | ||
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<img src="https://static.igem.org/mediawiki/2016/c/c7/T--Wageningen_UR--resultsvesicles.jpg"> | <img src="https://static.igem.org/mediawiki/2016/c/c7/T--Wageningen_UR--resultsvesicles.jpg"> | ||
| − | <figcaption>Figure | + | <figcaption>Figure 1. (a) The fluorescence of a two solutions with BBMVs obtained from <i>T. molitor</i> incorporated with fluorophores was measured over time. One in the presence and one in the absence of Cry3Aa. (b) The reaction rate constants for 6 individual measurements were calculated with the equation: fluorescence<sub>t</sub>=fluorescence<sub>(t=∞)</sub>-fluoresence<sub>(t=∞)</sub>∙e<sup>(-k∙t)</sup>+fluorescence<sub>(t=0)</sub>. </figcaption> |
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Revision as of 11:50, 18 October 2016
Results
In this section we like to present you the main results of the BeeT project. BeeT is engineered to produce a toxin specific for Varroa destructor, produce the toxin on the right time and incapable of escaping the hive alive. To accomplish this, we performed multiple experiments and created different models. The outcome is in short shown on this page.
In order to improve on existing methods, BeeT should effect Varroa mites only. To accomplish this we decided to make use of Cry toxins. A functional Cry toxin is only effective when specific binding occurs to the gut membrane of the target organism. Hereafter, the Cry toxins will form pores into the cell membrane, which results in cell death. As cell death occurs, the gut membrane becomes porous. Consequently, the organism dies. 1 To find a Cry toxin active against V. destructor we engineered our own toxins and we searched in nature for one as well.
Due to the parasitic nature of Varroa mites, testing Cry toxins proved to be very problematic. To overcome this problem we developed an in vitro test for Cry toxins. Out of the membranes of the target organism, brush border membrane vesicles (BBMVs) were made and incorporated with 6-carboxyfluorescein. A functional Cry toxins will create pores into the BBMVs, which then results in the leaking of fluorophores out of the BBMVs. Due to self-quenching behaviour of 6-carboxyfluorescein, this can be measured as an increase in fluorescence. As a proof of principle, BBMVs from the gut of Tenebrio molitor were made and loaded with 6-carboxyfluorescein to test the pore formation ability of Cry3Aa, which is known to be toxic to T. molitor larvae 3.
Figure 1a shows how the fluorescence increases of BBMVs incorporated with fluorophores in the presence and absence of Cry3Aa. A kinetic value could be coupled to this process. These values for multiple measurements for BBMVs in the presence and absence of Cry3Aa are shown in Figure 1b. From this can be concluded that the presence of a functional Cry protein results can be measured.
Here all the nice results regarding Cry Toxins be shown.
Here all the nice results regarding the ribo switch, toggle switch, metabolic modelling, and quorum sensing be shown.
Here all the nice results regarding the light kill switch and biocontainment will be shown.
Testing BeeT in a Beehive BEEHAVE
Beehave model and a nice conclusion
