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

Design

CPP-PYY Fusion Protein Design

A fusion protein that combines the main product peptide YY with signaling peptide, cell penetrating peptide and a thrombin-cleavable linker is designed. These additional parts all have individual functions.

1. Signaling peptide: Give bacteria the signal to secret Healthin after producing it. At the present stage, the signaling peptide for E.coli is used, however our ultimate goal is to express this fusion protein in Lactobacillus casei and another different signaling peptide is needed.
2. Cell penetrating peptide: Carry the whole fusion protein across membrane burden. The first discovered and the most widely studied cell penetrating peptide, TAT peptide, is used in this design. Its amino acid sequence is GRKKRRQRRRPQ.
3. Peptide YY: The main hormone in appetite controlling. For more detail, please check project description.
4. Thrombin cleavable linker: The linker is composed of the following amino acids: LEAGCKNFFPRSFTSCGSLE. The two Cysteine will bind together with a disulfide bind, and a dithiocyclopeptide linker is formed. This linker design can be adapted to diverse recombinant fusion proteins where in vivo separation of protein domains is required to achieve an improved therapeutic effect, and a desirable pharmacokinetic profile and biodistribution, of the functional domain.

Nisin Selection

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.[1] Researchers find that NisI seems to play a more crucial role in nisin immunity than the NisF/E/G complex.[2] Through experiments, either of each expressing in the heterologous bacteria is able to protect the host cells.[1] Moreover, the expression of nisI in Lactobacillus plantarum was assessed to be at the same level as in Lactococcus lactis.[2]

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

We introduced the part in the iGEM biobricks, NucA, as our main suicide gene. NucA codes for the mature form of nuclease from Staphylococcus aureus. This secreted enzyme is 5’-phosphodiesterase, which means it can cleave either single- or double-stranded DNA or RNA; therefore, it plays an vital role in the programmed cell death that involves DNA and RNA degradation.[3]

The problem encountered was that we hope our host cells alive in the product, while want them to die after producing moderate quantities of PYY in human intestines; also, when they are evacuated, back to the outside environment, the suicide gene must be turn on. We later searched the iGEM biobricks and found CI repressor that can bind to its regulated promoter, pCI, to repress the transcription.[4][5]

Now that the suicide gene NucA is inhibited by CI, the amount of CI protein inside the bacteria comes out to be immensely significant. To elaborate, we ought to carefully control the yield of CI produced by Lactobacillus casei ATCC393. The excess can make sure the cells are vigorous so that our main CPP-PYY gene circuit can function properly; on the other hand, the lack will turn on the transcription of thermonuclease which leads to the death of the host cells. To well control the quantity of CI repressor, we introduced the promoter of lac operon of Lactobacillus casei ATCC393 which is regulated by the ratio of lactose and glucose.

References

References

[1] 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)

[2] 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)

[3] Yu Hua, Jianghong Mengb, Chunlei Shia, Kirstin Hervina, Pina M. Fratamicoc, Xianming Shia. Characterization and comparative analysis of a second thermonuclease from Staphylococcus aureus. Microbiological Research, Volume 168, Issue 3, 30 March 2013, P.174–182. U.S.A. (2012)

[4] iGEM Part: http://parts.igem.org/Part:BBa_C0051

[5] iGEM Part: http://parts.igem.org/Part:BBa_R0051