Part:BBa_K1913007:Design
434- and lambda cI operon for tuning protein balance
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1898
Illegal BamHI site found at 1144 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
Design Notes
The 434 cI encoding gene in this part is (purposefully) not preceded by an RBS. In our research, a library of RBS's was inserted upstream of the 434 cI gene. This was done through 'round the horn PCR with the following primers: 5'-AAGGGTAATTATATAGTVHAGGRGGTTTAAGatgagtatttcttccagggtaa- 3' (Capital letters indicate the inserted RBS region) 5'-gctagcccaaaaaaacgggtatg- 3' We used primers with phosphorylated 5' ends for blunt-end ligation. The RBS region was kindly designed by Daniel Gerngross using the RedLibs1 algorithm. Be careful when ordering primers, the long primer can be very expensive. As a cheaper alternative, one could consider dividing the overhang over the two primers and phosphorylating 5' ends oneself.
The 434 cI protein encoded in this part contains a C-terminal LVA tag causing more rapid protein degradation, the lambda cI gene in this part does not encode an LVA tag (although BBa_K081007 does contain an LVA tag). Assembly was done through two rounds of Gibson Assembly. The LVA tag encoded by the lambda cI gene was purposefully left out of this part to provide different turnover rates for both cI proteins. This allow regulating the balance between the proteins based on changes in promoter strength over time.
Source
This part was Gibson assembled from fragments originating from biobrick parts BBa_I0500, BBa_C0052, BBa_K081007 and the pSB1C3 backbone.
References
1. Jeschek, M., Gerngross, D., & Panke, S. (2016). Rationally reduced libraries for combinatorial pathway optimization minimizing experimental effort. Nature Communications, 7, 11163. https://doi.org/10.1038/ncomms11163