Difference between revisions of "Part:BBa K538004:Design"

Revision as of 15:41, 16 September 2011

CspC (Polaribacter irgensii)

CspC K538004

CspC was used by team [http://2011.igem.org/Team:Amsterdam Amsterdam 2011] to create CryoBricks; cold resistance BioBricks. It encodes the CspC protein of Polaribacter irgensii, a homolog to E. coli's endogenous CspA and CspD. It's highly similar to CspA protein, which was also submitted to the registry. (BBa_K538003) See also the Main Page, or the team's [http://2011.igem.org/Team:Amsterdam/Project/Description Project Description] for further information.

Design Notes

The [http://www.ncbi.nlm.nih.gov/nuccore/88803076?from=235891&to=236082&report=gbwithparts sequence] of Polaribacter irgensii 's CspC protein was published by Murray et al. after shotgun sequencing its entire genome.[http://www.ncbi.nlm.nih.gov/nuccore/NZ_AAOG01000002] Team [http://2011.igem.org/Team:Amsterdam Amsterdam 2011] selected it for use in their CryoBricks following Uh et al. publishing they rescued a cold-sensitive E. coli by overexpressing this protein, greatly enhancing its freeze/thaw cycle survival rate. Pre- and suffixes were added to the sequence 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 1 SapI restriction site. Positions 73 through 79 matched SapI's target sequence: GAAGAGC. The site was recoded by changing the A on position 75 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

1. Murray et al. Direct submission to the NCBI database by the Craig Venter Institute, dated 2006/02/23
2. Uh et al. Rescue of a cold-sensitive mutant at low temperatures by cold shock proteins from Polaribacter irgensii KOPRI 22228, J. of Microbiol. 48 (6), 798-802 (2010)

Sequence and Features