Difference between revisions of "Part:BBa K5291037:Design"

(→‎Design Notes)
(→‎References)
 
(5 intermediate revisions by 2 users not shown)
Line 1: Line 1:
  
 
__NOTOC__
 
__NOTOC__
−
<partinfo>BBa_K5291999 short</partinfo>
+
<partinfo>BBa_K5291037 short</partinfo>
  
−
<partinfo>BBa_K5291999 SequenceAndFeatures</partinfo>
+
<partinfo>BBa_K5291037 SequenceAndFeatures</partinfo>
  
  
 
===Design Notes===
 
===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.
+
We replace the promoter of <i>sok</i> by P<sub>opdH</sub>, a promoter able to sense the concentration of citrate, and replace the promoter of <i>hok</i>/<i>mok</i> by PcW, a common weak constitutive promoter in <i>Pseudomonas aeruginosa</i>. When the bacteria are in the mangrovem, the relatively high concentration of citrate is able to activate P<sub>opdH</sub> and <i>sok</i> can be expressed to antagonize the effect of <i>hok</i>/<i>mok</i> due to the weak function of PcW. When the bacteria are out of the mangroves, P<sub>opdH</sub> cannot come into effect and the expression of <i>sok</i> is terminated, which enables <i>hok</i> to kill the cells.<br>
 +
When the plasmid with <i>hok</i>/<i>sok</i> system is lost, both of the genes cannot be expressed. However, the half-life of the <i>hok</i> mRNA is longer than the one of the <i>sok</i> antisense RNA, which leads to the result that the <i>hok</i> mRNA still exists when all the <i>sok</i> antisense RNA is degraded. The rest <i>hok</i> mRNA can encode toxin to kill the cell, achieving the function of plasmid anti-loss.
  
 
===Source===
 
===Source===
  
−
hok/mok, sok, Bi tern: Escherichia coli
+
P<sub>opdH</sub>, PcW: <i>Pseudomonas aeruginosa</i> PAO1<br>
−
PcW, PopdH: Pseudomonas aeruginosa PAO1
+
<i>sok</i>, <i>hok</i>/<i>mok</i>, BiTerm: <i>Escherichia coli</i>
  
 
===References===
 
===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.
+
[1]Puskas, L.G., et al., A periplasmic, alpha-type carbonic anhydrase from Rhodopseudomonas palustris is essential for bicarbonate uptake. Microbiology (Reading), 2000. 146 ( Pt 11): p. 2957-2966.<br>
 +
[2]Thorbecke, R., et al., The gene dosage effect of carbonic anhydrase on the biosynthesis of poly ( 3-hydroxybutyrate ) under autotrophic and mixotrophic culture conditions. POLYMER JOURNAL, 2021. 53(1): p. 209-213.<br>
 +
[3]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.

Latest revision as of 13:54, 27 September 2024


pAB-hok/sok


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 146
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 16
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We replace the promoter of sok by PopdH, a promoter able to sense the concentration of citrate, and replace the promoter of hok/mok by PcW, a common weak constitutive promoter in Pseudomonas aeruginosa. When the bacteria are in the mangrovem, the relatively high concentration of citrate is able to activate PopdH and sok can be expressed to antagonize the effect of hok/mok due to the weak function of PcW. When the bacteria are out of the mangroves, PopdH cannot come into effect and the expression of sok is terminated, which enables hok to kill the cells.
When the plasmid with hok/sok system is lost, both of the genes cannot be expressed. However, the half-life of the hok mRNA is longer than the one of the sok antisense RNA, which leads to the result that the hok mRNA still exists when all the sok antisense RNA is degraded. The rest hok mRNA can encode toxin to kill the cell, achieving the function of plasmid anti-loss.

Source

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

References

[1]Puskas, L.G., et al., A periplasmic, alpha-type carbonic anhydrase from Rhodopseudomonas palustris is essential for bicarbonate uptake. Microbiology (Reading), 2000. 146 ( Pt 11): p. 2957-2966.
[2]Thorbecke, R., et al., The gene dosage effect of carbonic anhydrase on the biosynthesis of poly ( 3-hydroxybutyrate ) under autotrophic and mixotrophic culture conditions. POLYMER JOURNAL, 2021. 53(1): p. 209-213.
[3]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.