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The future of agriculture is clearly connected with the production of food, feed, biomaterials, bioenergy, rare components like biopharmaceuticals and enzymes. Avery important aspect is the "unknown", which means the production of goods which we are not familiar with today. This brings up the subject of GMOs. The obvi­ous questions are: do we need GMOs and what kind of GMOs are necessary - GM plants, GM microorganisms, or GM animals? We have to recognize the different ways of using GMOs: direct consumption (food), indirect consumption (feed), raw materials (energy, biomaterials), and valuable components (like enzymes). The effects of GMOs are very different; the most visible one is the effect on the economy. There are also social, legal, environmental, international as well as religious, and mental effects that are very important. These "effects" are closely related to potential "dan­gers", both real (documented and reproducible) and imaginative (invented and not documented). To sum up, I would like to state the following: There is no way to avoid genetic engineering, and bioeconomy is the key to the future. However, we all have the right and privilege of free choice.
Cry4Aa 678 amino acids fragment (60 kDa) was cloned into Escherichia coli. After induction with IPTG the 60 kDa Cry4Aa fragment was purified by Ni chromatography, separated by SDS PAGE and identified by mass spectrometry (MS/MS). The 60 kDa Cry4Aa fragment exhibited high toxicity towards Ae. aegypti larvae. The earlier results [1] show that Pichia pastoris yeast cells expressing tmfA (synthetic gene coding for the Trypsin Modulating Oostatic Factor of Ae. aegypti) together with E. coli cells expressing Bti toxin genes (cry4Aa, cry11Aa, cyt1Aa and p20) are synergistic. Therefore, P. pastoris, which synthesizes high amounts of heterologous proteins was genetically engineered to produce TMOF and Cry4Aa. Codon-optimized synthetic genes, cry4Aa-tmfA, gst-cry4Aa-tmfA, tmfA and gfptmfA that were expressed by P. pastoris and fed to Ae. aegypti larvae caused 90% mortality. GST (glutathione-S-transferase) enhanced the activity of Cry4Aa-TMOF and protected it from heat denaturation and GFP (Green Fluorescent Protein)- TMOF allowed us to follow yeast cells consumption by individual larva using fluorescent microscopy. This report shows for the first time that 60 kDa Cry4Aa and TMOF expressed together in P. pastoris are highly toxic to Ae. aegypti larvae.
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