Team:NTU-LIHPAO-Taiwan/Project

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• Overview
  • Pigout / Stay in Shape
• Background
  • CPP-PYY
  • Nisin Selection
  • Suicide
• Design
  • CPP-PYY
  • Nisin Selection
  • Suicide
• Result
  • CPP-PYY
  • Nisin Selection
  • Suicide

Lactobacillus casei

Overview

Pigout Versus Stay in Shape

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 Lactobacillus casei , with cell penetrating peptides which contain large developmental potential in oral peptide drugs. We hope that we can make Lactobacillus casei secret CPP-PYY complex which can be another newly created oral peptide drugs.

Background

CPP-PYY

Cell Penetrating Peptide (CPP)

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.

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. 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.

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.

Peptide YY (PYY)

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.

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. 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.

Nisin Selection

Lactic Acid Bacteria (LAB)

In our project, we choose Lactobacillus casei 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.

Nisin

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 Lactococcus lactis, 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.

Suicide

Programmed Cell Death

The prototype of our product is an oral capsule which can function well while it reaches the human intestines. Although Lactobacillus casei 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.

Design

CPP-PYY

Nisin Selection

(Fig. Promoter-RBS-nisI-Ter)

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.

Suicide

Result

CPP-PYY

Nisin Selection

(Fig. Promoter-RBS-nisI-Ter)

(Fig. plasmid)

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 Lactobacillus plantarum was assessed to be at the same level as in Lactococcus lactis. [6]

The figure above shows our gene circuit for nisin selection. The promoter we chose was pUO19 from Escherichia coli which is also functional in Lactobacillus casei and the gene nisI helps Lactobacillus casei transform from nisin-sensitive into nisin-resistant. The fraction enlarged was latter proceeded ligation with CPP-PYY circuit, enabling the following selection.

Suicide

(Fig. plac-RBS-RFP-Ter)

(Fig. plac-RBS-CI-Ter)

(Fig. pCI-RBS-NcuA-Ter)