Team:NTU-LIHPAO-Taiwan/Design

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• Background
  • CPP-PYY
  • Nisin Selection
  • Suicide
• Design
  • CPP-PYY
  • Nisin Selection
  • Suicide
• Result
  • CPP-PYY
  • Nisin Selection
  • Suicide

Design

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

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.[6] 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.[7]

(Fig. promoter-RBS-NucA-Ter)

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.[8][9]

(Fig. pCI-RBS-NucA-Ter)&(cI)

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.

(part--alive)

(part--dead)

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