Team:NTU-LIHPAO-Taiwan/Parts

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• Prototype
  • Prototype
• Main Part
  • Part:BBa_K1841001
  • Part:BBa_K1841004
  • Part:BBa_K1841007
• Test Part
  • RFP
  • His-PYY
  • Signal peptide-PYY-His
  • Y2R
• References
  • References

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.

[Fig.1-1] Designed Plasmid

Main Part

Part: BBa_K1841001

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.

[Fig.2-1] Nisin Selection

Part: BBa_K1841004

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]

[Fig.2-2-1] Suicide Sense Part

[Fig.2-2-2] Suicide Kill Part

Part: BBa_K1841007

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]

[Fig.2-3] Cell penetrating peptide + PYY

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

RFP

[Fig.3-1] Suicide sense test

In this part, we use the RFP protein to testify the function of the lactose operon.

His-PYY

[Fig.3-2] 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

[Fig.3-3] Signal peptide + PYY

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

[Fig.3-4] Y2R

In this part, we use the constitutive promoter BBa_J23100 to produce Y2R. This part is constructed to verify the function of PYY.

References

References

[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

[4] C. W. le Roux and S. R. Bloom. Peptide YY, appetite and food intake.(2005)

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