Difference between revisions of "Part:BBa K864050:Design"

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===Design Notes===
 
===Design Notes===
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This ori was created by cloning pSC101ts from pSIM5 and making a V56A mutation in repA.  
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This ori was created by cloning pSC101ts from pSIM5[1] and making a V56A mutation in repA to revert the thermosensitivity. Another point mutation was made to remove one illegal SpeI site. According to a study of the replication of pSC101 by Yamaguchi and Masamune[2], the SpeI site is located in one of the repeat sequences (5') that are important for maintaining normal copy number of the plasmid. To minimize the effect of the mutagenesis needed to remove the SpeI site, the different repeat sequences were compared, and a mutation that would not disrupt the consensus sequence was chosen.
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Interestingly, the mutagenesis done in the pSB4X5 series of pSC101 origins to remove the SpeI site is made in such a way that the consensus repeat sequence is disrupted. This might be one of the reasons that the copy number of the pSB5X5-backbones are higher than expected.
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===Source===
 
===Source===
  
−
The pSC101ts origin was cloned from the pSIM5 plasmid from Datta et al, see reference below. One point mutation was made to remove one illegal SpeI site.
+
The pSC101ts origin was cloned from the pSIM5 plasmid from Datta et al, see reference below.
  
 
===References===
 
===References===
 
[http://dx.doi.org/10.1016/j.gene.2006.04.018] Datta, S., Costantino, N. & Court, D.L. A set of recombineering plasmids for gram-negative bacteria. Gene 379, 109–115 (2006).
 
[http://dx.doi.org/10.1016/j.gene.2006.04.018] Datta, S., Costantino, N. & Court, D.L. A set of recombineering plasmids for gram-negative bacteria. Gene 379, 109–115 (2006).
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[http://www.springerlink.com/content/v88817g163j5jwh7/] Yamaguchi K., Masamune Y. Autogenous regulation of synthesis of the replication protein in plasmid pSC101. Mol. Gen. Genet. 200, 362–367 (1985).

Latest revision as of 00:13, 30 September 2012

pSC101 low copy origin of replication


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2130
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This ori was created by cloning pSC101ts from pSIM5[1] and making a V56A mutation in repA to revert the thermosensitivity. Another point mutation was made to remove one illegal SpeI site. According to a study of the replication of pSC101 by Yamaguchi and Masamune[2], the SpeI site is located in one of the repeat sequences (5') that are important for maintaining normal copy number of the plasmid. To minimize the effect of the mutagenesis needed to remove the SpeI site, the different repeat sequences were compared, and a mutation that would not disrupt the consensus sequence was chosen. Interestingly, the mutagenesis done in the pSB4X5 series of pSC101 origins to remove the SpeI site is made in such a way that the consensus repeat sequence is disrupted. This might be one of the reasons that the copy number of the pSB5X5-backbones are higher than expected.

Source

The pSC101ts origin was cloned from the pSIM5 plasmid from Datta et al, see reference below.

References

[http://dx.doi.org/10.1016/j.gene.2006.04.018] Datta, S., Costantino, N. & Court, D.L. A set of recombineering plasmids for gram-negative bacteria. Gene 379, 109–115 (2006).

[http://www.springerlink.com/content/v88817g163j5jwh7/] Yamaguchi K., Masamune Y. Autogenous regulation of synthesis of the replication protein in plasmid pSC101. Mol. Gen. Genet. 200, 362–367 (1985).