Part:BBa_K3861026

pKF02 (pKF01 + PlldR-sicP-sptP-Pep8)

The lactate-inducible lldR promoter (BBa_K1847008) fused to RBS(SicP)-sicP-sptP (BBa_K3861028) and Pep8, inserted in the plasmid backbone pKF01. SicP-SptP is the secretion signal and chaperone for secretion of a POI though the SPI-1 T3SS of Salmonella Typhimurium.¹ SptP167 is shortened to the first 167 amino acids that contain the secretion signal and the sicP binding domain needed for functional secretion. SicP is the SptP specific chaperone that unfolds the POI secondary structure for SPI-1 T3SS mediated export.² Pep8 is an artificial peptide that was shown to play a potential role in the re-induction of p53 translation which leads to decreased tumor growth in mice xenograft.¹ p53 acts as tumor suppressor by preserving the integrity of the genome. p53 loss of function is associated with more than half of all human cancers.^2,3 Further, Pep8 was shown to sensitize cancer cells to an anticancer drug doxorubicin.³ we transformed the plasmid into a thyA deletion Salmonella Typhimurium background. This mutation is required since the complementation of thyA acts as maintenance marker to keep the plasmid in the bacteria. After this, the growth medium of strain background no longer needed to be supplement with thymine. We were also able to isolate the plasmid from this strain in order to sequence the plasmid. The sequencing showed correct results. Furthermore, the PlldR promoter was repressed by LldR, encoded on the plasmid.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 3012
  Illegal NheI site found at 3035
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 997
  Illegal XhoI site found at 2804
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

1. Lee, S. H. & Galán, J. E. Salmonella type III secretion-associated chaperones confer secretion-pathway specificity. Mol. Microbiol. 51, 483–495 (2004).
2. Stebbins, C. E. & Galán, J. E. Maintenance of an unfolded polypeptide by a cognate chaperone in bacterial type III secretion. Nature 414, 77–81 (2001).
3. Lucchesi, C. A., Zhang, J., Ma, B., Chen, M. & Chen, X. Disruption of the Rbm38-eIF4E Complex with a Synthetic Peptide Pep8 Increases p53 Expression. Cancer Res. 79, 807–818 (2019).
4. Vogelstein, B., Lane, D. & Levine, A. J. Surfing the p53 network. Nature 408, 307–310 (2000).
5. Vousden, K. H. & Prives, C. Blinded by the Light: The Growing Complexity of p53. Cell 137, 413–431 (2009).