Part:BBa_K891234:Design
D168A Double Cysteine Mutant of Variola Virus Topoisomerase
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 895
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 895
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 895
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 895
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 895
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
This part needs to be placed behind an inducible promoter, since it is toxic to the cell and its own plasmid.
Source
Dr. Hwang first cloned the wild type topoisomerse sequence from the Variola virus genome as demonstrated in "Regulation of Catalysis by the Smallpox Virus Topoisomerase". This part is expressed in a pet29a vector. The two cysteines from the wild type strain have been mutated out and the 168th amino acid has been changed from aspartic acid to alanine. A his tag has been added to the end of this sequence.
References
Hwang, Young, Nana Minkah, Kay Perry, Gregory D. Van Duyne, and Frederic D. Bushman. "Regulation of Catalysis by the Smallpox Virus Topoisomerase -- Hwang Et Al. 281 (49): 38052 -- Journal of Biological Chemistry." Regulation of Catalysis by the Smallpox Virus Topoisomerase -- Hwang Et Al. 281 (49): 38052 -- Journal of Biological Chemistry. Journal of Biological Chemistry, 10 Oct. 2006. Web. 03 Oct. 2012. <http://hwmaint.jbc.org/cgi/content/full/281/49/38052>.