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

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<div class="Text1">Overview</div>
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<div class="Text1">Prototype</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">Prototype</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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The iGEM Team NTU-LIHPAO-Taiwan 2015 had built a new biological system for the iGEM community. The original design of the system making up the (Product) contains the three elements listed below, in a probiotic – Lactobacillus casei ATCC 393.
 
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<div class="Text1">Background</div>
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<div class="Text1">Main Part</div>
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<div class="Text2" id="Second1">CPP-PYY</div>
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<div class="Text2" id="Second1">Part: BBa_xxxxxxx</div>
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<div class="Text3"><div class="Text_underline">Cell Penetrating Peptide (CPP)</div></div>
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<div class="Text3"><div class="Text_underline">nisI</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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Research from Torsten et al. revealed that the highest level of acquired nisin tolerance was achieved after coordinated expression of all four nisin immunity genes, containing nisI, nisF, nisE, and nisG. [1] Functional analyses provided evidence that NisI acts as a nisin-sequestering protein and that NisFEG acts as a nisin exporter that expels nisin molecules from the cytoplasmic membrane into the environment.
 
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<div class="Text2" id="Second1">Part: BBa_xxxxxxx</div>
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<div class="Text3"><div class="Text_underline">plac promoter</div></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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Lactose operon of Lactobacillus casei In Lactobacillus casei ATCC393 [pLZ15–], the lactose genes are grouped in a cluster transcribed as single operon. The cluster lacTEGF encodes an antiterminator protein (LacT), lactose-specific elements (LacE and LacF) of the phosphotransferase system (PTS) and a phospho-β-galactosidase (LacG).
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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.  
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The promoter region contains a cre element (catabolite responsive element) overlapping the –35 region, which is followed by a highly conserved sequence, the ribonucleic antiterminator (RAT) sequence, and a terminator structure.  
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The antiterminator activity of LacT is also negatively controlled by glucose, possibly by PTS-mediated phosphorylation as explained below [2].
 
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<div class="Text2" id="Second1">Part: BBa_xxxxxxx</div>
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<div class="Text3"><div class="Text_underline">TAT-PYY</div>
 
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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.  
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TAT can bring the big molecule substance like DNA、protein、peptide to penetrate the cell. Usually, named as protein transduction domain or membrane transduction domain, it is less than 40 amino acid[3]. PYY belongs to the gastrointestinal hormones. Once the intestine detects the nutrition, the epidermal cell, L cell of ileum and colon will secrete the PYY.While the blood pass the hypothalamus, PYY will bind to the neuropeptide Y receptor in ventromedial nuclei causing the sense of satiation[4].
 
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<div class="Text3"><div class="Text_underline">Peptide YY (PYY)</div></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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In this part, we use the constitutive promoter BBa_J23100 to produce signal peptide-pyy-histidine fusion protein. With the his-tag, we can easily purify the signal peptide-pyy from other non-his-tag protein. This part is constructed to test the amount of the pyy that can secret out of the L. casei.
 
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<div class="Text1">Test Part</div>
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<div class="Text2" id="Second1">Gfp</div>
 
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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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In this part, we use the gfp protein to testify the function of the lactose operon.
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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="Text2" id="Second1">His-pyy</div>
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<div class="Text2" id="Second2">Nisin Selection</div>
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<div class="Text3"><div class="Text_underline">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 are able to metabolite carbohydrates to produce lactic acid with the yield over 50%. [1] 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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In this part, we use the constitutive promoter BBa_J23100 to produce histidine-pyy fusion protein. With the his-tag, we can easily purify the pyy from other non-his-tag protein. This part is constructed to test the amount of the pyy under the constitutive promoter in the L. casei.
 
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<div class="Text3"><div class="Text_underline">Nisin</div></div>
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<div class="Text2" id="Second2">Signal peptide-pyy-his</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). [2] 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. [3] 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 Design section.
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In this part, we use the constitutive promoter BBa_J23100 to produce signal peptide-pyy-histidine fusion protein. With the his-tag, we can easily purify the signal peptide-pyy from other non-his-tag protein. This part is constructed to test the amount of the pyy that can secret out of the L. casei.
 
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<div class="Text2" id="Second2">Y2r</div>
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<div class="Text2" id="Second3">Suicide</div>
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<div class="Text3"><div class="Text_underline">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 NcuA. [4] It is a thermonuclease that degrades both plasmid and chromosomal DNA. For more information, please go to the Design section.
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In this part, we use the constitutive promoter BBa_J23100 to produce y2R. This part is constructed to verify the function of pyy.
 
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<div class="Text1">Reference</div>
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<div class="Text1">Design</div>
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<div class="Text2" id="Third1">CPP-PYY</div>
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<div class="Text2" id="Second3">Reference</div>
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<div class="Text2" id="Third2">Nisin Selection</div>
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<div class="Text3">(Fig. Promoter-RBS-nisI-Ter)</div>
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Studies have showed that for nisin resistance, the immunity lipoprotein NisI as well as the ABC transporter-homologous system NisF/E/G is involved. Functional analysis suggests that NisI acts as nisin-intercepting protein, while NisF/E/G complex acts as exporter that expels the unwanted nisin molecules from cytoplasm to the outer environment. [4] Researchers find that NisI seems to play a more crucial role in nisin immunity than the NisF/E/G complex. [5] Through experiments, either of each expressing in the heterologous bacteria is able to protect the host cells.
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[1] Torsten, S., Stefan, H., Irina, S., and K. D. Entian. Function of Lactococcus lactis Nisin Immunity Genes nisI and nisFEG after Coordinated Expression in the Surrogate Host Bacillus subtilis. The Jurnal of Biological Chemistry, USA. , Vol. 278, pp. 89 -94 (2003)
 
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<div class="Text2" id="Third3">Suicide</div>
 
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<div class="Text1">Result</div>
 
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<div class="Text2" id="Fourth1">CPP-PYY</div>
 
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<div class="Text2" id="Fourth2">Nisin Selection</div>
 
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<div class="Text3">(Fig. Promoter-RBS-nisI-Ter)</div>
 
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<div class="Text3">(Fig. plasmid)</div>
 
 
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Studies have showed that for nisin resistance, the immunity lipoprotein NisI as well as the ABC transporter-homologous system NisF/E/G is involved. Functional analysis suggests that NisI acts as nisin-intercepting protein, while NisF/E/G complex acts as exporter that expels the unwanted nisin molecules from cytoplasm to the outer environment. [5] Researchers find that NisI seems to play a more crucial role in nisin immunity than the NisF/E/G complex. [6] Through experiments, either of each expressing in the heterologous bacteria is able to protect the host cells. [5] Moreover, the expression of nisI in <i>Lactobacillus plantarum</i> was assessed to be at the same level as in <i>Lactococcus lactis</i>. [6]
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[2] Yu-Kuo Tsai, Hung- Wen Chen, Ta-Chun Lo and Thy-Hou Lin. Specific point mutation in Lactobacillus casei ATCC 27139 cause the phenotype switch from Lac- to Lac+. Microbiology, pp. 751-760(2009)
 
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The figure above shows our gene circuit for nisin selection. The promoter we chose was pUO19 from <i>Escherichia coli</i> which is also functional in <i>Lactobacillus casei</i> and the gene <i>nisI</i> helps <i>Lactobacillus casei</i> transform from nisin-sensitive into nisin-resistant. The fraction enlarged was latter proceeded ligation with CPP-PYY circuit, enabling the following selection.
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[3] Ju-Chen Cheng, 2009, Investigation on the Characteristic of Heparin-Binding Haemagglutinin 3 Adhesin (HBHA)-Related Peptides and their Transcytosis
 
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<div class="Text2" id="Fourth3">Suicide</div>
 
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<div class="Text3">(Fig. plac-RBS-RFP-Ter)</div>
 
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<div class="Text3">(Fig. plac-RBS-CI-Ter)</div>
 
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<div class="Text3">(Fig. pCI-RBS-NcuA-Ter)</div>
 
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Revision as of 11:06, 14 August 2015

NTU-LIHPAO-Taiwan

Lactobacillus casei
Prototype
Prototype
The iGEM Team NTU-LIHPAO-Taiwan 2015 had built a new biological system for the iGEM community. The original design of the system making up the (Product) contains the three elements listed below, in a probiotic – Lactobacillus casei ATCC 393.
Main Part
Part: BBa_xxxxxxx
nisI
Research from Torsten et al. revealed that the highest level of acquired nisin tolerance was achieved after coordinated expression of all four nisin immunity genes, containing nisI, nisF, nisE, and nisG. [1] Functional analyses provided evidence that NisI acts as a nisin-sequestering protein and that NisFEG acts as a nisin exporter that expels nisin molecules from the cytoplasmic membrane into the environment.
Part: BBa_xxxxxxx
plac promoter
Lactose operon of Lactobacillus casei In Lactobacillus casei ATCC393 [pLZ15–], the lactose genes are grouped in a cluster transcribed as single operon. The cluster lacTEGF encodes an antiterminator protein (LacT), lactose-specific elements (LacE and LacF) of the phosphotransferase system (PTS) and a phospho-β-galactosidase (LacG). The promoter region contains a cre element (catabolite responsive element) overlapping the –35 region, which is followed by a highly conserved sequence, the ribonucleic antiterminator (RAT) sequence, and a terminator structure. The antiterminator activity of LacT is also negatively controlled by glucose, possibly by PTS-mediated phosphorylation as explained below [2].
Part: BBa_xxxxxxx
TAT-PYY
TAT can bring the big molecule substance like DNA、protein、peptide to penetrate the cell. Usually, named as protein transduction domain or membrane transduction domain, it is less than 40 amino acid[3]. PYY belongs to the gastrointestinal hormones. Once the intestine detects the nutrition, the epidermal cell, L cell of ileum and colon will secrete the PYY.While the blood pass the hypothalamus, PYY will bind to the neuropeptide Y receptor in ventromedial nuclei causing the sense of satiation[4].
In this part, we use the constitutive promoter BBa_J23100 to produce signal peptide-pyy-histidine fusion protein. With the his-tag, we can easily purify the signal peptide-pyy from other non-his-tag protein. This part is constructed to test the amount of the pyy that can secret out of the L. casei.
Test Part
Gfp
In this part, we use the gfp protein to testify the function of the lactose operon.
His-pyy
In this part, we use the constitutive promoter BBa_J23100 to produce histidine-pyy fusion protein. With the his-tag, we can easily purify the pyy from other non-his-tag protein. This part is constructed to test the amount of the pyy under the constitutive promoter in the L. casei.
Signal peptide-pyy-his
In this part, we use the constitutive promoter BBa_J23100 to produce signal peptide-pyy-histidine fusion protein. With the his-tag, we can easily purify the signal peptide-pyy from other non-his-tag protein. This part is constructed to test the amount of the pyy that can secret out of the L. casei.
Y2r
In this part, we use the constitutive promoter BBa_J23100 to produce y2R. This part is constructed to verify the function of pyy.
Reference
Reference
[1] Torsten, S., Stefan, H., Irina, S., and K. D. Entian. Function of Lactococcus lactis Nisin Immunity Genes nisI and nisFEG after Coordinated Expression in the Surrogate Host Bacillus subtilis. The Jurnal of Biological Chemistry, USA. , Vol. 278, pp. 89 -94 (2003)
[2] Yu-Kuo Tsai, Hung- Wen Chen, Ta-Chun Lo and Thy-Hou Lin. Specific point mutation in Lactobacillus casei ATCC 27139 cause the phenotype switch from Lac- to Lac+. Microbiology, pp. 751-760(2009)
[3] Ju-Chen Cheng, 2009, Investigation on the Characteristic of Heparin-Binding Haemagglutinin 3 Adhesin (HBHA)-Related Peptides and their Transcytosis