Difference between revisions of "Part:BBa K535005:Design"
(New page: __NOTOC__ <partinfo>BBa_K535005 short</partinfo> <partinfo>BBa_K535005 SequenceAndFeatures</partinfo> ===Design Notes=== Some codons of the original ''Clostridium acetobutylicum'' ATCC...) |
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| − | Both genes are part of ''Clostridium acetobutylicum'' ATCC 824 chromosome, some modifications to the natural sequence were made, for more details check out the “Design Notes” section. Ferredoxins' NCBI accession number [http://www.ncbi.nlm.nih.gov/protein/NP_346944 NP_346944]; Hydrogenase's accession number [http://www.ncbi.nlm.nih.gov/protein/AAB03723 AAB03723]. | + | Both genes are part of ''Clostridium acetobutylicum'' ATCC 824 chromosome, some modifications to the natural sequence were made, for more details check out the “Design Notes” section. Ferredoxins' NCBI accession number [http://www.ncbi.nlm.nih.gov/protein/NP_346944 NP_346944]; Hydrogenase's NCBI accession number [http://www.ncbi.nlm.nih.gov/protein/AAB03723 AAB03723]. |
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===References=== | ===References=== | ||
*Christina M Agapakis, Daniel C Ducat, Patrick M Boyle, Edwin H Wintermute, Jeffrey C Way, Pamela A Silver (2010) Insulation of a synthetic hydrogen metabolism circuit in bacteria. Journal of Biological Engineering 4:3. | *Christina M Agapakis, Daniel C Ducat, Patrick M Boyle, Edwin H Wintermute, Jeffrey C Way, Pamela A Silver (2010) Insulation of a synthetic hydrogen metabolism circuit in bacteria. Journal of Biological Engineering 4:3. | ||
Latest revision as of 01:18, 26 September 2011
HydA1 - FeOx construction (Hydrogenase-ferredoxin fussion)
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 793
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1321
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 327
Illegal NgoMIV site found at 1581
Illegal NgoMIV site found at 1728 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2203
Illegal BsaI.rc site found at 1446
Design Notes
Some codons of the original Clostridium acetobutylicum ATCC 824 HydA1 and FeOx sequences have been changed for synonimous ones according to the Codon Adaptation Index (CAI) procedure in order to optimize its expression and to optimize Rhizobium etli CFN42 fitness as well.
The CAI indicates how similar the Codon Usage (CU) in a coding sequence (CDS) is to that of highly/constitutively expressed genes. It is not a cause of high gene expression, but it is necessary to optimize resource usage. To optimize a sequence according to the CAI procedure we first obtained relative adaptiveness (w) for each codon (1.- most frequent codon. 0.- non-existent codon) in R. etli and then we substitute codons in target CDS with all synonymous codons with greatest w.
The fusion between HydA1 and FeOx is mediated by a 14aa long glycine/serine rich linker. The whole construction transcription is mediated by a Rhizobium etli CFN42 nifH promoter which is active under the nitrogen fixation conditions which include low oxygen concentration.
We added at the end a double TAA terminator.
Unwanted restriction sites had been changed for synonimous codons.
Source
Both genes are part of Clostridium acetobutylicum ATCC 824 chromosome, some modifications to the natural sequence were made, for more details check out the “Design Notes” section. Ferredoxins' NCBI accession number [http://www.ncbi.nlm.nih.gov/protein/NP_346944 NP_346944]; Hydrogenase's NCBI accession number [http://www.ncbi.nlm.nih.gov/protein/AAB03723 AAB03723].
References
- Christina M Agapakis, Daniel C Ducat, Patrick M Boyle, Edwin H Wintermute, Jeffrey C Way, Pamela A Silver (2010) Insulation of a synthetic hydrogen metabolism circuit in bacteria. Journal of Biological Engineering 4:3.