Team:NTU-LIHPAO-Taiwan/Description

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Motivation

Motivation

We notice the obesity problem raising people’s concern worldwide. A research in 2014 shows that around 39% of adults are overweight and 13% are obese. One of the reasons lies behind is the out-of-controlled appetite. People could not stop eating and become obese. Then they go on a diet but fail to tolerate hunger. Some people would then turn to diet pills for help, but ends up suffering from side effects.

We provide a healthier and easier method to solve the obesity problem.We design a fusion protein, “Healthin”, aiming to lower obese people’s appetite. As for the bacteria we use, Lactobacillus casei is chosen rather than E.coli because Lactobacillus casei are harmless probiotics which ameliorate human’s health. Thus, it is obviously our best choice for carrying the oral drug, “Healthin”. The product “Healthin” is based on the peptide YY which our body use to lower appetite after food intake. Also, some special peptides are added to transport peptide YY from bacteria to its working place and function well. We believe these could work through synthetic biology and ultimately make it to help people move back to healthy diet quicker and easier.

Background

CPP-PYY

Cell Penetrating Peptide (CPP)

CPP is a kind of short segment peptide that can carry macromolecules such as DNA, proteins, and peptides to penetrate cell membrane. In general, CPPs are relatively short peptides that consist of less than 40 amino acids and are able to enter cells by means of various mechanisms, including endocytosis, and are further able to assist in the intracellular delivery of covalently or noncovalently conjugated bioactive cargos.[1]

The first discovered and extensively studied CPP is TAT peptide, which derived from human immunodeficiency virus-1 (HIV-1). Later, more CPPs were discovered and researches even started to synthesize CPPs like R9. Some examples of CPPS with their physical characters were listed in table 1 below.[2]

There are two major cell penetrating mechanism: energy-independent direct penetration and energy-consuming endocytosis. In these two pathways are three similar steps: membrane interaction, membrane permeation, secretion of CPPs to cytoplasm.

1. Direct penetration: First of all, positively charged CPPs draw negatively charged components of membrane such as HS, phospholipid di-layer. Then, integral proteins fold. Consequently, the membrane temporarily collapses and inverted micelles or pores may appear. CPPs could later penetrate cell membrane and reach the cytoplasm. This increases partial hydrogen concentration and forms the concentration gradient, which enables CPPs to move along. However, the final location of CPPs depends on their concentration and the properties of their cargo.
2. Endocytosis : Several pathways such as pinocytosis, macropinocytosis, receptor-mediated endocytosis are included. Vesicles are formed after CPPs interacts with membrane to transport them into cytoplasm. Past studies considered direct penetration as major penetrating mechanism. Nonetheless, current studies show that endocytosis more or less involves in the penetrating process of different CPPs in various conditions.

However, specific description remains unknown. Different CPPs, CPP-cargo complexes, secondary structures, and even experimental conditions all have effect on penetrating pathways.

Peptide YY (PYY)

Peptide YY is a gastrointestinal (GI) hormone that can restrain our appetite. Because this short peptide’s the terminal amino acids in each end are tyrosine (Y), it is named peptide YY (PYY). There are two main endogenous forms: PYY_1–36 and PYY_3–36.[3] Both are stable without the need of disulfide bonds. The latter is produced by the action of the enzyme dipeptidyl peptidase-IV (DPP-IV).[4] The conformational change of PYY3-36 alleviates its binding affinity to the NPY Y2 receptor (Y2R).

Peptide YY plays an important role in controlling appetite. After our intestine absorbs micromolecule nutrient, the L-type endocrine cell (L cell) will secret PYY to blood. Along the blood flow, PYY could enter the arcuate nucleus in hypothalamus and then bind with Y2R. The binding decreases our appetite and inhibits eating.[5]

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 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.

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).[1] 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.[2] 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. It is a thermonuclease that degrades both plasmid and chromosomal DNA. For more information, please go to the [Design] section.

Reference

Reference

[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. Antimicrob Agents Chemother, Vol. 27, No. 5, p.841–845. Germany. (1985)

[2] Cleveland J et al. Bacteriocins: safe, natural antimicrobials for food preservation. Int J Food Microbiol, Vol.71, p.1-20. USA.(2001)

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