Part:BBa_K5291037:Design

BCD1 bicistronic design

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 622
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 622
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 24
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 622
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 622
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 496
  Illegal BsaI.rc site found at 419
  Illegal SapI site found at 30
  

Design Notes

The hok/sok system and BiTerm are from Escherichia coli but we have found that they are effective in engineered Pseudomonas aeruginosa. And we use promoters PcW and PopdH native in P. aeruginosa to ensure the effect of gene expression.

Source

hok/mok, sok, Bi tern: Escherichia coli PcW, PopdH: Pseudomonas aeruginosa PAO1

References

[1]Bielecki, P., et al., Cross talk between the response regulators PhoB and TctD allows for the integration of diverse environmental signals in Pseudomonas aeruginosa. Nucleic Acids Res, 2015. 43(13): p. 6413-25. [2]Tamber, S., et al., Characterization of OpdH, a Pseudomonas aeruginosa porin involved in the uptake of tricarboxylates. J Bacteriol, 2007. 189(3): p. 929-39. [3]Brocker, M., et al., Citrate utilization by Corynebacterium glutamicum is controlled by the CitAB two-component system through positive regulation of the citrate transport genes citH and tctCBA. J Bacteriol, 2009. 191(12): p. 3869-80. n[4]Underhill, S. and M.T. Cabeen, Redundancy in Citrate and cis-Aconitate Transport in Pseudomonas aeruginosa. J Bacteriol, 2022. 204(12): p. e0028422. [5]Kikuchi, Y., et al., Correlation between the spread of IMP-producing bacteria and the promoter strength of bla(IMP) genes. J Antibiot (Tokyo), 2024. 77(5): p. 315-323. [6]Gerdes, K., et al., Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of the E. coli relB operon. The EMBO Journal, 1986. 5(8): p. 2023-2029. [7]Gerdes, K., et al., The hok killer gene family in gram-negative bacteria. The New biologist, 1990. 2: p. 946-56. [8]Gong, C.C. and S. Klumpp, Modeling sRNA-Regulated Plasmid Maintenance. PLoS One, 2017. 12(1): p. e0169703. [9]Gerdes, K. and E.G. Wagner, RNA antitoxins. Curr Opin Microbiol, 2007. 10(2): p. 117-24. [10]Thisted, T. and K. Gerdes, Mechanism of post-segregational killing by the hok/sok system of plasmid R1: Sok antisense RNA regulates hok gene expression indirectly through the overlapping mok gene. Journal of Molecular Biology, 1992. 223(1): p. 41-54. [11]Franch, T., A.P. Gultyaev and K. Gerdes, Programmed cell death by hok/sok of plasmid R1: Processing at the hok mRNA 3′-end triggers structural rearrangements that allow translation and antisense RNA binding11Edited by D. E. Draper. Journal of Molecular Biology, 1997. 273(1): p. 38-51. [12]Gong, C.C. and S. Klumpp, Modeling sRNA-Regulated Plasmid Maintenance. PLoS One, 2017. 12(1): p. e0169703. [13]Chen YJ, Liu P, Nielsen AA, Brophy JA, Clancy K, Peterson T, Voigt CA. Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat Methods. 2013 Jul;10(7):659-64. doi: 10.1038/nmeth.2515. Epub 2013 Jun 2. PMID: 23727987.