Part:BBa_K3788004:Design
6His-P20, Flag-Cry11Aa and Strep-Cyt1Aa coding sequence
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 154
Illegal XhoI site found at 2438 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
After the sequence eventual mutation was prevented : Amino acids have been changed to avoid the formation of a stop codon and also to remove restriction sites corresponding to RFC10 and RFC25.
Creating the BBa_K3788004 part
As for the cloning of the part BBa_K3788004 into the plasmid pBAD24 MSC3, the following strategy was thought. The plasmid pBAD24_6hisp20_flagcry11Aa (BBa_K3788003 part into pBAD24 vector) can be used after a digested with KpnI restriction enzyme and used to insert the gene strepcyt1Aa (BBa_K3788000) by recombination by the SLIC cloning method.
Cloning strategy table : Thus, strepcyt1Aa (BBa_K3788000) has to be amplified by PCR with the specifics primers to be able to insert strepcyt1Aa gene into pBAD24_6hisp20_flagcry11Aa .
Source
The 6His_P20__Flag_cry11Aa__Strep_cyt1Aa sequence is made from BBa_K3788002, BBa_K3788001 (BBa_K3788003) and BBa_K3788000 parts.
Theses 2 parts are optimised sequences for E. coli obtain from Part:BBa_K2938008, Part:BBa_K2938005 and Part:BBa_K2938003.
References
- Bravo, A., Gill, S. S. & Soberón, M. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49, 423–435 (2007).
- Hua, G., Masson, L., Jurat-Fuentes, J. L., Schwab, G. & Adang, M. J. Binding Analysis of Bacillus thuringiensis Cry δ-Endotoxins Using Brush Border Membrane Vesicles of Ostrinia nubilalis. Appl. Environ. Microbiol. 67, 872–879 (2001).
- Manasherob, R. et al. Effect of Accessory Proteins P19 and P20 on Cytolytic Activity of Cyt1Aa from Bacillus thuringiensis subsp. israelensis in Escherichia coli. Curr. Microbiol. 43, 355–364 (2001).