Difference between revisions of "Part:BBa K1218011:Design"

Revision as of 17:19, 13 October 2016

Cas9

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1642
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 3921
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 4863
Illegal BsaI.rc site found at 4840

Design Notes

Mutagenesis to remove internal EcoRI site

Cas9 for chloroplast

Sequence was codon optimized with software developed at UCL in Saul Purton's lab working on algal biotechnology. Can be found at the following link: https://www.ucl.ac.uk/algae/Genetic_engineering_tools. The sequence was additionally modified to remove all Bsa1 and BsmBI sites (illegal for phytobrick syntax) in the coding sequence and where possible the codon maintained as well as the genetic bias.

No direct source for part, need to be synthesized, sequence based on S. pyrogenes (http://www.uniprot.org/uniprot/Q99ZW2). Due to the large size of the protein, iDT synthesized part in 4 pieces which we were able to assemble through golden gate with the appropriate primers to maintain the coding sequence intact. Alternatively, Gibson could have been used for assembly.

Source

CRISPR-Cas system from streptococcus pyogenes; cloned from plasmids obtained from Bikard et al.

References

http://nar.oxfordjournals.org/content/41/15/7429 David Bikard, Wenyan Jiang, Poulami Samai, Ann Hochschild, Feng Zhang3, and Luciano A. Marraffini. (2013) Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Research, 41 (15), 7429-7437.

@@ Line 10: / Line 10: @@
 ===Cas9 for chloroplast===
-Sequence was codon optimized with software developed at UCL in Saul Purton's lab working on algal biotechnology. Can be found at the following link: https://www.ucl.ac.uk/algae/Genetic_engineering_tools.
+Sequence was codon optimized with software developed at UCL in Saul Purton's lab working on algal biotechnology. Can be found at the following link: https://www.ucl.ac.uk/algae/Genetic_engineering_tools. The sequence was additionally modified to remove all Bsa1 and BsmBI sites (illegal for phytobrick syntax) in the coding sequence and where possible the codon maintained as well as the genetic bias.
-The sequence was additionally modified to remove all Bsa1 and BsmBI sites (illegal for phytobrick syntax) in the coding sequence and where possible the codon maintained as well as the genetic bias.
 No direct source for part, need to be synthesized, sequence based on <i>S. pyrogenes</i> (http://www.uniprot.org/uniprot/Q99ZW2). Due to the large size of the protein, iDT synthesized part in 4 pieces which we were able to assemble through golden gate with the appropriate primers to maintain the coding sequence intact. Alternatively, Gibson could have been used for assembly.