Part:BBa_K2127001:Design
Inducible High Expression His-Tagged Photosystem II CP47 subunit
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 658
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 782
Illegal AgeI site found at 1379
Illegal AgeI site found at 1583 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1103
Illegal SapI site found at 1780
Design Notes
BBa_K2127001 was designed to be expressible in E. coli to allow for easier characterization and study of the Transcription Factor Binding site (TFBS) obtained from Synechocystis PCC 6803. As shown by Dr MA with the study on promotor Cpc560, the TFBS showed a distinct effect on the ability to upregulate protein expression of a gene under promotor control. To see about developing a viable high expression and inducible promotor in for cyanobacterium, this TFBS was combined with a lac promotor under LacI regulation. The construct was then combined with our characterized His tagged psbB gene to determine and analyze the expression levels of the CP47His subunit. The construct was then combined with double stop codons and our own derivative of the BBa_B0015 double terminator (BBa_K2127007) to ensure that only the subunit will be expressed.
To test the construct, E coli DH5α F' Iq was chosen as a suitable transformation and expression host.This is due to the ease of transformation into this host and the presence of the mutated strong lac repressor. The idea of the system is to facilitate the future work involving photosystem components within well characterized organisms such as E coli. Consequentially, we also aim to develop a way to create inducible systems within cyanobacteria that will allow for easier genetic and protein work.
Source
Organism: Synechocystis spp PCC6803