Part:BBa_K2212004:Design
GLA w/ pconII+RiboswitchF+DYKDDDDK+rrnBT1
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 526
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 415
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 41
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 380
Design Notes
The part is intended to be used in Synechococcus elongatus PCC 7942. Therefore, the promoter and terminator are all common construct that have been used in the Golden Lab. To prevent excess expression of the part proteins having negative effects in Synechococcus elongatus PCC 7942 cells, a reboswitch F was added to make the transcription controllable. Riboswitch F was used because it was found by the Golden Lab to have the best alternation effect. Codon optimization for the coding sequence used Synechocystis sp. PCC6803 as target species because the tool used did not have option for Synechococcus elongatus PCC 7942. Therefore, the available option Synechocystis sp. PCC6803 that is closes to S. elongatus PCC 7942 was used. The protein coding sequence was originated from plant Saccharopolyspora erythraea because its cellular conditions best resemble that of Synechococcus elongatus PCC 7942, of which has pH 7-8 and temperature 30-40 degree Celsius.
Source
The gene sequence was taken from Kegg gene and cross checked using BLAST protein. The gene sequence was then codon optimized for Synechocystis sp. PCC6803 using the Integrated DNA Technologies Inc.'s online codon optimization tool. The sequence for promoter pconII, riboswitch F, rrnB T1 terminator sequence, DYKDDDDK flag tag sequence, and filler sequence were taken from database of the Golden Lab.
References
Amy T. Ma, Calvin M. Schmidt and James W. Golden. Regulation of Gene Expression in Diverse Cyanobacterial Species Using Theophylline-Responsive Riboswitches. Appl. Environ. Microbiol. May 2017 83:10 e00035-17
Taton A, Lis E, Adin DM, Dong G, Cookson S, Kay SA, et al. (2012) Gene Transfer in Leptolyngbya sp. Strain BL0902, a Cyanobacterium Suitable for Production of Biomass and Bioproducts. PLoS ONE7(1): e30901.
Post, D.A. & Luebke, V.E. Purification, cloning, and properties of α-galactosidase from Saccharopolyspora erythraea and its use as a reporter system. Appl Microbiol Biotechnol (2005) 67: 91.