Difference between revisions of "Part:BBa K2916006:Design"
 
(4 intermediate revisions by the same user not shown)
Line 8: Line 8:
 
===Design Notes===
 
===Design Notes===
  
−
The sequence was extracted from the pET21a-IleRS-His plasmid and expressed in the BL21 (DE3) E.coli strain.
+
The sequence was extracted from the pET21a-LysRS-His plasmid and expressed in the BL21 (DE3) E.coli strain.
  
−
Codon optimization : 1179 from G to C
+
Codon optimization : 1245 from A to G
  
−
Codon optimization : 1179 from G to C
+
Codon optimization : 1344 from T to A
  
−
Codon optimization : 1179 from G to C  
+
Codon optimization : 480 from C to G
  
−
Second stop codon was added in compliance with iGEM standards.  
+
Second stop codon was added in compliance with iGEM standards.
  
 
===Source===
 
===Source===
Line 25: Line 25:
  
 
===References===
 
===References===
 +
 +
<i>
 +
McClain, William H. “Rules That Govern TRNA Identity in Protein Synthesis.” Journal of Molecular Biology, vol. 234, no. 2, Nov. 1993, pp. 257–80. DOI.org (Crossref), doi:10.1006/jmbi.1993.1582.
  
 
Cell-free translation reconstituted with purified components. Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T. Nat Biotechnol. 2001 Aug;19(8):751-5. doi: 10.1038/90802. 10.1038/90802 PubMed 11479568
 
Cell-free translation reconstituted with purified components. Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T. Nat Biotechnol. 2001 Aug;19(8):751-5. doi: 10.1038/90802. 10.1038/90802 PubMed 11479568
  
−
Lavickova, Barbora, and Sebastian J. Maerkl. A Simple, Robust, and Low-Cost Method to Produce the PURE Cell - Free System. preprint, Synthetic Biology, 18 Sept. 2018. DOI.org (Crossref), doi:10.1101/420570.
+
Lavickova, Barbora, and Sebastian J. Maerkl. A Simple, Robust, and Low-Cost Method to Produce the PURE Cell - Free System. preprint, Synthetic Biology, 18 Sept. 2018. DOI.org (Crossref), doi:10.1101/420570.</i>

Latest revision as of 02:01, 22 October 2019


LysRS protein equipped with a 6x HIS affinity tag


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1121
    Illegal XhoI site found at 1516
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 475


Design Notes

The sequence was extracted from the pET21a-LysRS-His plasmid and expressed in the BL21 (DE3) E.coli strain.

Codon optimization : 1245 from A to G

Codon optimization : 1344 from T to A

Codon optimization : 480 from C to G

Second stop codon was added in compliance with iGEM standards.

Source

Sebastian Maerkl Lab at EPFL, Switzerland.

http://www.addgene.org/124114/

References

McClain, William H. “Rules That Govern TRNA Identity in Protein Synthesis.” Journal of Molecular Biology, vol. 234, no. 2, Nov. 1993, pp. 257–80. DOI.org (Crossref), doi:10.1006/jmbi.1993.1582.

Cell-free translation reconstituted with purified components. Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T. Nat Biotechnol. 2001 Aug;19(8):751-5. doi: 10.1038/90802. 10.1038/90802 PubMed 11479568

Lavickova, Barbora, and Sebastian J. Maerkl. A Simple, Robust, and Low-Cost Method to Produce the PURE Cell - Free System. preprint, Synthetic Biology, 18 Sept. 2018. DOI.org (Crossref), doi:10.1101/420570.