Difference between revisions of "Part:BBa K538002:Design"

Latest revision as of 10:24, 16 September 2011

SheDnaK (Shewanella sp. Ac10)

SheDnaK K538002

SheDnaK was used by team [http://2011.igem.org/Team:Amsterdam Amsterdam 2011] to create CryoBricks; cold resistance BioBricks. It encodes the DnaK of Shewanella sp. Ac10, a homolog of E. coli 's own DnaK that's active at lower temperatures. See also the Main Page, or the team's [http://2011.igem.org/Team:Amsterdam/Project/Description Project Description] for further information.

Design Notes

Prefacing Yoshimune et al. 's publication regarding SheDnaK 's activity relative to that of E. coli 's endogenous DnaK[http://www.springerlink.com/content/f50n0gahppxacytb/], this protein's [http://www.ncbi.nlm.nih.gov/nuccore/AY728897.1 sequence] was published. Pre- and suffixes were added to this as clarified in OpenWetWare's [http://openwetware.org/wiki/Biobrick_standard BioBrick standards] and, as is recommended, the TAA stop codon was replaced with TAATAA. The sequence's conformity with Assembly standard 10 and putative future standards was ensured using the [http://bioweb2.pasteur.fr/docs/EMBOSS/recoder.html EMBOSS recoder], by recoding restriction sites of EcoRI, XbaI, SpeI, PstI, NotI, PvuII, XhoI, AvrII, NheI and SapI. Note that to ensure compatibility with [http://dspace.mit.edu/handle/1721.1/46747 RFC 21] and [http://dspace.mit.edu/handle/1721.1/45140 RFC 25], an additional 4 restriction sites must be recoded. For RFC 21 compatibility, no BamHI or BglII sites may be present in the sequence. Likewise, for RFC 25, no AgeI or NgoMIV sites may occur.

[http://bioweb2.pasteur.fr/docs/EMBOSS/recoder.html EMBOSS recoder] detected and silently mutated 5 different restriction sites.

Positions 344 through 349 matched PvuII's target sequence: CAGCTG. The site was recoded by changing the A on position 346 to a G.
Positions 1280 through 1285 also matched PvuII's target sequence. The site was recoded by changing the A on position 1282 to a G.
Positions 724 through 729 matched EcoRI's target sequence: GAATTC. The site was recoded by changing the A on position 726 to a G.
Positions 945 through 950 matched XbaI's target sequence: TCTAGA. The site was recoded by changing the T on position 945 to a G.
Positions 1669 through 1674 matched NheI's target sequence: GCTAGC. The site was recoded by changing the T on position 1671 to a G.

Source

De novo synthesis by [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/gene-synthesis/GeneArt-Gene-Synthesis.html GeneArt]

References

Yoshimune et al. Cold-active DnaK of an Antarctic psychrotroph Shewanella sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures, Extremophiles 9 (2), 145-150 (2005)

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 747
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 11: / Line 11: @@
 Prefacing Yoshimune ''et al.'' 's publication regarding ''SheDnaK'' 's activity relative to that of ''E. coli'' 's endogenous ''DnaK''[http://www.springerlink.com/content/f50n0gahppxacytb/], this protein's [http://www.ncbi.nlm.nih.gov/nuccore/AY728897.1 sequence] was published. Pre- and suffixes were added to this as clarified in OpenWetWare's [http://openwetware.org/wiki/Biobrick_standard BioBrick standards] and, as is recommended, the TAA stop codon was replaced with TAATAA. The sequence's conformity with [https://parts.igem.org/Help:Assembly_standard_10 Assembly standard 10] and putative future standards was ensured using the [http://bioweb2.pasteur.fr/docs/EMBOSS/recoder.html EMBOSS recoder], by recoding restriction sites of ''EcoRI'', ''XbaI'', ''SpeI'', ''PstI'', ''NotI'', ''PvuII'', ''XhoI'', ''AvrII'', ''NheI'' and ''SapI''. Note that to ensure compatibility with [http://dspace.mit.edu/handle/1721.1/46747 RFC 21] and [http://dspace.mit.edu/handle/1721.1/45140 RFC 25], an additional 4 restriction sites must be recoded. For RFC 21 compatibility, no ''BamHI'' or ''BglII'' sites may be present in the sequence. Likewise, for RFC 25, no ''AgeI'' or ''NgoMIV'' sites may occur.
+[http://bioweb2.pasteur.fr/docs/EMBOSS/recoder.html EMBOSS recoder] detected and silently mutated 5 different restriction sites.
+* Positions 344 through 349 matched PvuII's target sequence: CAGCTG. The site was recoded by changing the A on position 346 to a G.
+* Positions 1280 through 1285 also matched PvuII's target sequence. The site was recoded by changing the A on position 1282 to a G.
+* Positions 724 through 729 matched EcoRI's target sequence: GAATTC. The site was recoded by changing the A on position 726 to a G.
+* Positions 945 through 950 matched XbaI's target sequence: TCTAGA. The site was recoded by changing the T on position 945 to a G.
+* Positions 1669 through 1674 matched NheI's target sequence: GCTAGC. The site was recoded by changing the T on position 1671 to a G.
 ===Source===