healthy, thin, and keep fit2015.igem.org/files/poster/NTU-LIHPAO-Taiwan.pdf · healthy, thin, and...
1
healthy, thin, and keep fit A food-grade selection method is required for our further use of Healthin in human beings. We choose nisin, a kind of bacteriocins, as our selection marker. Studies have showed that the immunity lipoprotein NisI is able to protect cell membrane from the pore-forming activity of nisin. We tried to simulate the pathways of designed L. casei and secreted CPP-PYY complex from oral ingestion to final PYY releasing. The ultimate goal is to build the general formula, and to find out the suitable and optimal dose for Healthin. The pathways consist of 5 parts : 1. Bacteria distribution 2. CPP-PYY penetration efficiency 3. Absorption into bloodstream 4. Thrombin cleavage efficiency 5. Healthin drug effect We applied MATLAB for calculation and simulation. Here we considered 10 9 of total L. casei intake, and 10 -10 μg/min of complex production rate per L. casei. The optimal concentration of PYY is 0.1 μg/L for appetite suppression ideally. The result of final concentration of PYY (μg/L) in the blood is shown below. Nisin Selection Modeling Background Fusion Protein Suicide Methodology 0 0.5 1 1.5 2 2.5 3 3.5 4 0 5 10 15 20 25 30 35 E.coli growth curve CONTROL absorbtion(A) SUICIDE absorbtion(A) To prevent environmental pollution and over-dosage, we have designed a suicide part to control death-time. General formula : [Final concentration of PYY in the blood (μg/L)] = 11745 × [Total L. casei intake] × [Distribution ratio of L. casei] × [CPP-PYY complex production rate per L. casei (μg/min)] 1. Ballantyne, G.H. Peptide YY(1-36) and peptide YY(3-36): Part I. Distribution, release and actions. Obes Surg. 2006; 16:651-8 2. Torsten Stein, Stefan Heinzmann, Irina Solovieva, and Karl-Dieter Entian. Function of Lactococcus lactis nisin immunity genes nisI and nisFEG after coordinated expression in the surrogate host Bacillus subtilis. The Journal of Biological Chemistry, Vol. 278, No. 1, Issue of January 3, p.89–94. Germany. (2003) 3. T. M. Takala ·P. E. J. Saris. A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI. Appl Microbiol Biotechnol 59, p.467–471. U.S.A. (2003) 4. 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) 5. Y. K. Lee and P. S. Ho et al. Permanent colonization by Lactobacillus casei is hindered by the low rate of cell division in mouse gut. Applied and Environmental Microbiology, Vol. 70, No. 2. Finland. (2003) 6. Liang JF and Yang VC. Insulin-cell penetrating peptide hybrids with improved intestinal absorption efficiency. Biochem Biophys Res Commun, Vol. 335, p. 734-738. USA. (2005) 1.9 million overweight adults in 2014 Obesity treatment market 15 billions Anti-obesity drug market 8.4 billions by2022 Peptide YY a GI hormone that can restrain our appetite. Its binding with receptor Y2R in hypothalamus decreases our appetite and inhibits eating. Healthin is a designed fusion protein included peptide YY and several auxiliary peptide. SP-PYY PYY TAT-PYY Y2R 0 2 4 6 8 10 12 14 16 18 0 0.5 1 1.5 2 2.5 3 3.5 4 10^7CFU/mL Nisin (logIU) Nisin Immunity Test protein purification secretion test penetration test binding test plasmid construction selection test suicide test functional test nisin damages nisI protection NucA damages suicide part control death-time test function efficiency death-time computation taken dose computation calculation Human Practice Our Team Acknowledgment WIKI http://2015.igem.org/Team:NTU-LIHPAO-Taiwan FB National Taiwan University iGEM Reference Each gene sequence of individual peptide is successfully assembled with the backbone pSB1C3 and pcDNA3. Gene sequencing results show the successful combination of each part and the receptor Y2R with the plasmid backbone pSB1C3 and pcDNA3. Street random survey Promoting health information on Facebook fan page Publicizing health information through holding conference Skype and meetup with team Oxford Small conference with team Mingdao Asia Meetup at NCTU
Transcript of healthy, thin, and keep fit2015.igem.org/files/poster/NTU-LIHPAO-Taiwan.pdf · healthy, thin, and...
healthy, thin, and keep fit
A food-grade selection method is required for our further use of Healthin in human beings. We choose nisin, a kind of bacteriocins, as our selection marker. Studies have showed that the immunity lipoprotein NisI is able to protect cell membrane from the pore-forming activity of nisin.
We tried to simulate the pathways of designed L. casei and secreted CPP-PYY complex from oral ingestion to final PYY releasing. The ultimate goal is to build the general formula, and to find out the suitable and optimal dose for Healthin. The pathways consist of 5 parts : 1. Bacteria distribution 2. CPP-PYY penetration efficiency 3. Absorption into bloodstream 4. Thrombin cleavage efficiency 5. Healthin drug effect
We applied MATLAB for calculation and simulation. Here we considered 109 of total L. casei intake, and 10-10 μg/min of complex production rate per L. casei. The optimal concentration of PYY is 0.1 μg/L for appetite suppression ideally. The result of final concentration of PYY (μg/L) in the blood is shown below.
Nisin Selection
Modeling
Background
Fusion Protein
Suicide
Methodology
0
0.5
1
1.5
2
2.5
3
3.5
4
0 5 10 15 20 25 30 35
E.coli growth curve
CONTROLabsorbtion(A)
SUICIDEabsorbtion(A)
To prevent environmental pollution and over-dosage, we have designed a suicide part to control death-time.
General formula : [Final concentration of PYY in the blood (μg/L)] = 11745 × [Total L. casei intake] × [Distribution ratio of L. casei] × [CPP-PYY complex production rate per L. casei (μg/min)]
1. Ballantyne, G.H. Peptide YY(1-36) and peptide YY(3-36): Part I. Distribution, release and actions. Obes Surg. 2006; 16:651-8 2. Torsten Stein, Stefan Heinzmann, Irina Solovieva, and Karl-Dieter Entian. Function of Lactococcus lactis nisin immunity genes nisI and nisFEG after coordinated
expression in the surrogate host Bacillus subtilis. The Journal of Biological Chemistry, Vol. 278, No. 1, Issue of January 3, p.89–94. Germany. (2003) 3. T. M. Takala · P. E. J. Saris. A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI. Appl Microbiol Biotechnol 59, p.467–471. U.S.A.
(2003) 4. 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) 5. Y. K. Lee and P. S. Ho et al. Permanent colonization by Lactobacillus casei is hindered by the low rate of cell division in mouse gut. Applied and Environmental
Microbiology, Vol. 70, No. 2. Finland. (2003) 6. Liang JF and Yang VC. Insulin-cell penetrating peptide hybrids with improved intestinal absorption efficiency. Biochem Biophys Res Commun, Vol. 335, p. 734-738.
USA. (2005)
1.9 million overweight adults in 2014
Obesity treatment market
15 billions
Anti-obesity drug market
8.4 billions by2022
Peptide YY a GI hormone that can restrain our appetite. Its binding with receptor Y2R in hypothalamus decreases our appetite and inhibits eating.
Healthin is a designed fusion protein included peptide YY and several auxiliary peptide.
SP-PYY PYY TAT-PYY Y2R
0
2
4
6
8
10
12
14
16
18
0 0.5 1 1.5 2 2.5 3 3.5 4
10
^7C
FU/m
L
Nisin (logIU)
Nisin Immunity Test
protein purification secretion test penetration test binding test
plasmid construction
selection test suicide test functional test
nisin damages nisI protection
NucA damages suicide part control death-time test
function efficiency death-time computation taken dose computation
calculation
Human Practice
Our Team
Acknowledgment
WIKI http://2015.igem.org/Team:NTU-LIHPAO-Taiwan FB National Taiwan University iGEM
Reference
Each gene sequence of individual peptide is successfully assembled with the backbone pSB1C3 and pcDNA3.
Gene sequencing results show the successful combination of each part and the receptor Y2R with the plasmid backbone pSB1C3 and pcDNA3.
Street random survey Promoting health information on Facebook fan page Publicizing health information through holding conference Skype and meetup with team Oxford Small conference with team Mingdao Asia Meetup at NCTU
