Composite

Part:BBa_K3924042

Designed by: Yuhan Yang   Group: iGEM21_Tsinghua   (2021-10-21)


PnorV-RiboJ

PnorV-RiboJ

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 198
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Profile

Name: PnorV-riboJ
Base Pairs:275bp
Origin: E.coli Nissle 1917
Properties: A sensor to nitric oxide with an often-used insulator.

Usage and Biology

In order to heal the intestinal tract damage, one of notable symptoms of IBD, we adopted a special therapy expressing the therapeutic proteins controllably by E.coli Nissle 1917 (EcN) in situ. The design is based on a ternary system: sensor - secretion peptide - therapeutic proteins.

Fig.1 General design of the treatment ternary system

The therapeutic protein secreted by the engineered bacteria needs to be strictly and delicately controlled to avoid any possible safety problems since the final working environment is the GI tract, and some therapeutic proteins are carcinogenic and may cause safety problems. To prevent this, we want to create several gut sensor parts as an intelligent switch to conditionally regulate the expression of therapeutic proteins.
PnorV is one of candidate sensors we screened out, with which we can create spatiotemporal expression of therapeutic protein based on demand.
Nitric oxide (NO) can be a general marker for inflammation and PnorV can bind and initiate downstream gene expression.
RiboJ, a self-splicing ribozyme, is a common used insulator. With a hairpin structure, it can reduce unexpected interactions between neighboring sequences in a genetic circuit.[1]Some reports also say insulation with RiboJ can increase downstream gene transcript abundance.[2]
Insertion of RiboJ behind the sensor part is likely to improve the performance of the sensors.
PnorV(BBa_K1153000) was designed by iGEM13_Kent, and RiboJ(BBa_K3424024) was designed by iGEM20_UFlorida .

Table.1 Improvement for previous sensors with RiboJ
BBa_K2507000
BBa_K2507001		 BBa_K2507006
BBa_K2507007		 BBa_K1153000 BBa_K3924040 BBa_K3924041 BBa_K3924042
thsS/R ttrS/R PnorV Pttr-RiboJ Pths-RiboJ PnorV-RiboJ

Design and Construction

PnorV was acquired from Nissle 1917 genome. While riboJ is an element from RGP plasmid. We used PCR and HiFi Assembly to link them together.

Reference

[1] Vlková, M., Morampalli, B. R., & Silander, O. K. (2021). Efficiency of the synthetic self-splicing RiboJ ribozyme is robust to cis- and trans-changes in genetic background. MicrobiologyOpen, 10(4), e1232.
[2] Clifton, K. P., Jones, E. M., Paudel, S., Marken, J. P., Monette, C. E., Halleran, A. D., Epp, L., & Saha, M. S. (2018). The genetic insulator RiboJ increases expression of insulated genes. Journal of biological engineering, 12, 23.


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