Difference between revisions of "Team:NTU-LIHPAO-Taiwan/Attributions"

 
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<span class="title">Overview</span>
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<span class="title">Attributions</span>
 
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<li><a href="#First1">Pigout Versus Stay in Shape</a></li>
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<li><a href="#First1">Attributions</a></li>
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<span class="title">Background</span>
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<li><a href="#Second1">CPP-PYY</a></li>
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<li><a href="#Second2">Nisin Selection</a></li>
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<div class="Text1">Attributions</div>
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<div class="Text2" id="First1">Pigout Versus Stay in Shape</div>
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<div class="Text2" id="First1">Dr. Chi-Chih Liao, the senior leader in Lihpao Life Science Corp.</div>
 
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This year, iGEM team of NTU-LIHPAO-Taiwan take note of the problem that the obesity condition in Taiwan has deteriorated. Moreover, the market is flooded with those unverified slimming drugs that harm the public health simultaneously. Therefore, we hope to initiate our project from the key peptide “Peptide YY (PYY)” which can control appetite, and take advantage of the probiotic characteristic of <i>Lactobacillus casei</i>, with cell penetrating peptides which contain large developmental potential in oral peptide drugs. We hope that we can make <i>Lactobacillus casei</i> secret CPP-PYY complex which can be another newly created oral peptide drugs.
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Giving us advice on culture techniques of <i>Lactococcus casei</i>, protocols of transformation of Lactococcus casei, providing lab materials.
 
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<div class="Text1">Background</div>
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<div class="Text2">Prof. Yen-Rong Chen</div>
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<div class="Text2" id="Second1">CPP-PYY</div>
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<div class="Text3"><div class="Text_TitleUnderline">Cell Penetrating Peptide (CPP)</div></div>
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CPP is a kind of short segment peptide that can spontaneously carry macromolecules such as DNA, proteins, and peptides to penetrate cell membrane. Generally speaking, they often contain less than 40 amino acids and also called protein transduction domain (PTDs) or membrane transduction domain (MTDs). CPP which is first discovered and extensively studied is TAT protein derived from human immunodeficiency virus-1 (HIV-1) and antennapedia homeodomain (Antp) transcription factor comes from Drosophila melanogaster. The shortest segments diagnosed from the sequences are TAT and penetratin, and more and more CPPs come into existence from the following studies. There are also synthetic amino acid sequences such as R9.
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Providing his lab for us to do experiments, helping with materials and equipment, offering advice on lab techniques.
 
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<div class="Text3"><div class="Text_TitleUnderline">Xin-Hong, Li, students of Chen’s lab</div></div>
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Helping us during our brainstorming, giving advice on protocol design ,teaching us lab technique.
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<div class="Text3"><div class="Text_TitleUnderline">Chao-Wei Hsu, students of Chen’s lab</div></div>
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Aiding protocol design, helping with result and data analysis, giving advice on new protocols for improving our experiment, helping with cell culture.
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<div class="Text3"><div class="Text_TitleUnderline">Teh-Wei Wang, students of Chen’s lab</div></div>
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Giving advice on protocol design, helping with result and data analysis, giving advice on new protocol for improving our experiment, teaching us cell culture techniques, helping with cell culture.
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<div class="Text3"><div class="Text_TitleUnderline">Hsing-Yi Yu, students of Chen’s lab</div></div>
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Providing protocols for protein extraction, teaching us how to use sonicator and lyophilizer.
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<div class="Text3"><div class="Text_TitleUnderline">Yi-Hao Lin, students of Chen’s lab</div></div>
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Providing HEK293 cells and advice on cell culture.
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<div class="Text2" id="First1">Prof. Li-Jiuan Shen, School of Pharmacy, National Taiwan University</div>
 
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However, the mechanism of CPP penetrating cell membrane is still vague, and different CPPs, CPP-cargo complexes, secondary structures have significant effect on distinct cells’ penetrating mechanism. Currently, there are majorly two pathways: energy-independent direct penetration and energy-consuming endocytosis, and two pathways both have three same steps: membrane interaction, membrane permeation, secreting CPPs to cytoplasm.
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Providing Caco-2 cells, information of cell penetrating peptide, protocols and advice on experiment of cell culture and transport assay.
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<li>Direct penetration : First of all, positively charged CPPs draw negatively charged molecules on membrane such as HS, phospholipid dilayer, with integral proteins folded causing membrane temporarily collapse making CPP penetrate cell membrane, and it may form an inverted micelle or a pore. It increases partial hydrogen concentration that CPPs enter the cytoplasm, forming the concentration gradient, it makes CPPs move from one side to the other, and whether TAT penetrate the cell depends on the concentration and cargo’s properties. </li>
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<li>Endocytosis : Endocytosis uses pinocytosis, macropinocytosis, receptor-mediated endocytosis to form vesicles, transporting CPPs into cytoplasm. Because in the early phase scientists recognized that CPP can penetrate cell membrane in 4oC so that they deducted CPP majorly penetrate cell membrane with direct penetration. Nonetheless, current studies show that endocytosis more or less involves in the penetrating process of different CPPs in various conditions.</li>
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<div class="Text3"><div class="Text_TitleUnderline">Peptide YY (PYY)</div></div>
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<div class="Text3"><div class="Text_TitleUnderline">Po-Chuan Chiu, students of Shen’s lab</div></div>
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Giving us protocols of Caco-2 cell culture and transport assay.
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<div class="Text2" id="First1">Prof. Nan-Wei Su, Department of Agricultural Chemistry, National Taiwan University</div>
 
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Peptide YY is a short peptide that can restrain our appetite. Because the peptide’s head and tail are both amino acid, tyrosine (Y), it is named peptide YY (PYY). PYY has two forms: PYY 1-36 is the unmodified form, and PYY 3-36 is the kind of PYY cut off two amino acids in N-terminal side by dipeptidyl peptidase-IV. Each contains 60% and 40% of all PYY.
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Providing Caco-2 cells, culture medium, protocols and advice on experiment of cell culture and transport assay, lending us Millicell ERS (Voltohmmeter).
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In the situation of PYY binding to the receptors, PYY 1-36’s affinity to Y1, Y2, Y4,and Y5 are all high. However, because PYY 3-36 is cut off two amino acids in N-terminal side causing conformational change, its affinity to Y2 is higher than others. Since both two types of PYY don’t require disulfide bond to stable its structure, it can spontaneously become a stable and activated form in the solution.
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PYY is classified as gastrointestinal(GI) hormone. After intestine absorbs micromolecule nutrients, ileum and colon epithelial cells will secret PYY to blood. As PYY contact hypothalamus by blood circulation.
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<div class="Text3"><div class="Text_TitleUnderline">Shan-Da Wang, students of Su’s lab</div></div>
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<div class="Text4">
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Helping us on transport assay, teaching us how to use Millicell ERS.
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<div class="Text2" id="Second2">Nisin Selection</div>
 
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<div class="Text3"><div class="Text_TitleUnderline">Lactic Acid Bacteria (LAB)</div></div>
 
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In our project, we choose <i>Lactobacillus casei</i> ATCC393 as the study material which belongs to the diverse family of lactic acid bacteria. Lactic acid bacteria are not a formal term in taxonomy; as a matter of fact, the lactic acid bacteria are referred to as a group of microorganism that is able to metabolite carbohydrates to produce lactic acid with the yield over 50%. One notable fact is that lactic acid bacteria are long be used in the manufacture of dairy products, and therefore they are generally regarded as safe (GRAS). Moreover, they are the most representative probiotics in the intestines.
 
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<div class="Text3"><div class="Text_TitleUnderline">Nisin</div></div>
 
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Since we are aim to produce PYY for the further use in human beings, a food-grade experimental procedure must be conducted. Here we choose nisin, a kind of bacteriocins excreted by <i>Lactococcus lactis</i>, as our selection marker. Bacteriocins are antimicrobial peptides produced by bacteria to kill or inhibit the growth of similar or closely related bacterial strain(s).<a href="#Reference1">[1]</a> For nisin, however, it can form pores in the bacterial cytoplasmic membrane and decrease the membrane potential; thus, it has a broader range of target cells.<a href="#Reference2">[2]</a> Nisin-producing organisms have their specific way to protect their cell membrane from the nisin pore-forming activity. We use this characteristic of nisin immunity to select cells containing out target plasmids. The detailed self-protection mechanism is discussed in the <a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Design"><b>Design</b></a> section.
 
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<div class="Text2" id="Second3">Suicide</div>
 
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<div class="Text3"><div class="Text_TitleUnderline">Programmed Cell Death</div></div>
 
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The prototype of our product is an oral capsule which can function well while it reaches the human intestines. Although <i>Lactobacillus casei</i> ATCC393 as a probiotic would not do harm to the consumers’ healthy, for safety concern, cell apoptosis should be introduced due to several reasons. To begin with, we want to control the quantity of PYY within a proper range so as not to cause side-effects. What we value the most is the yield of PYY produced by the time the cell died; therefore, the concentration of bacteria being put into the capsule can be determined with its penetration rate taken into consideration. Another critical reason is that the genes transfer among bacteria and with the environment must be diminished. Otherwise, it may not only interfere the gut flora, but also contaminate the surroundings. With those concerns, we found a desirable part in the iGEM biobricks, namely <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159105">NcuA</a>. It is a thermonuclease that degrades both plasmid and chromosomal DNA. For more information, please go to the Design section.
 
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<div class="Text1">Reference</div>
 
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<div class="Text2" id="Third1">Reference</div>
 
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[1] E Ruhr and H G Sahl. Mode of action of the peptide antibiotic nisin and influence on the membrane potential of whole cells and on cytoplasmic and artificial membrane vesicles. <i>Antimicrob Agents Chemother</i>, Vol. 27, No. 5, p.841–845. Germany. (1985)
 
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<div class="Text3" id="Reference2">
 
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[2] Cleveland J <i>et al</i>. Bacteriocins: safe, natural antimicrobials for food preservation. <i>Int J Food Microbiol</i>, Vol.71, p.1-20. USA.(2001)
 
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Latest revision as of 13:11, 18 September 2015

NTU-LIHPAO-Taiwan

Attributions
Dr. Chi-Chih Liao, the senior leader in Lihpao Life Science Corp.
Giving us advice on culture techniques of Lactococcus casei, protocols of transformation of Lactococcus casei, providing lab materials.
Prof. Yen-Rong Chen
Providing his lab for us to do experiments, helping with materials and equipment, offering advice on lab techniques.
Xin-Hong, Li, students of Chen’s lab
Helping us during our brainstorming, giving advice on protocol design ,teaching us lab technique.
Chao-Wei Hsu, students of Chen’s lab
Aiding protocol design, helping with result and data analysis, giving advice on new protocols for improving our experiment, helping with cell culture.
Teh-Wei Wang, students of Chen’s lab
Giving advice on protocol design, helping with result and data analysis, giving advice on new protocol for improving our experiment, teaching us cell culture techniques, helping with cell culture.
Hsing-Yi Yu, students of Chen’s lab
Providing protocols for protein extraction, teaching us how to use sonicator and lyophilizer.
Yi-Hao Lin, students of Chen’s lab
Providing HEK293 cells and advice on cell culture.
Prof. Li-Jiuan Shen, School of Pharmacy, National Taiwan University
Providing Caco-2 cells, information of cell penetrating peptide, protocols and advice on experiment of cell culture and transport assay.
Po-Chuan Chiu, students of Shen’s lab
Giving us protocols of Caco-2 cell culture and transport assay.
Prof. Nan-Wei Su, Department of Agricultural Chemistry, National Taiwan University
Providing Caco-2 cells, culture medium, protocols and advice on experiment of cell culture and transport assay, lending us Millicell ERS (Voltohmmeter).
Shan-Da Wang, students of Su’s lab
Helping us on transport assay, teaching us how to use Millicell ERS.
Maintained by the iGEM team NTU-LIHPAO-Taiwan    ©2015 NTU-LIHPAO-Taiwan