Part:BBa_K3788004
6His-P20, Flag-Cry11Aa and Strep-Cyt1Aa coding sequence
The Part BBa_K3788004 is a part composed with BBa_K3788003 (6His_P20__Flag_cry11Aa) and BBa_K3788000 (Strep_cyt1Aa). Theses 3 proteins are Bacillus thuringiensis proteins. Cry11Aa has a Flag tag in N-ter, Cyt1Aa has a strep tag II in N-ter and P20 has a 6his tag in N-ter to allow purification of the proteins and specific detection by Western blot.
The gene was optimised for E. coli K12. To allow the expression in operon, an RBS sequence is added before the ATG of Flag_cry11Aa gene sequence and Strep_cyt1Aa gene sequence.
Expected sizes :
| Lengths of DNA sequence | Proteins sizes | |
|---|---|---|
| 6his-P20 | 570 bp | 20 kDa |
| Flag-Cry11Aa | 1956 bp | 70 kDa |
| Strep-Cyt1Aa | 777 bp | 28 kDa |
Mode of action of Cry toxins:
Cry toxins, in the form of a pro-toxin (inactive), will undergo proteolytic cleavage at their N-terminus end, allowing the unmasking of the zone of interaction with the receptor. Mature Cry toxins recognize glycoprotein-type receptors on the surface of midgut cells, more specifically the microvilli of the apical membranes. The II and III domains, forming 3 β-sheets, allow this interaction. Once recognized,the domain I, forming 7 amphipathic α-helices, one of which is hydrophobic, is inserted into the membrane to allow the formation of the pre-pore. From this first step emerges the formation of Cry toxins oligomers and the formation of the pore. This allows ion leakage and cell lysis. Following this, an event cascade lead to sepsis resulting in the death of the insect
Cry11Aa has a toxic action, specific to A. aegypti because its -8 loop, region -4 and domain II loop 3 recognize the mosquito midgut BBMV receptor.
Mode of action of Cyt toxins:
Cyt (Cytolitic) toxins are produced in pro-toxin form, they will undergo proteolytic cleavage in N-ter and C-ter allowing the release of the active form of Cyt toxins. The mature forms will interact with the membrane lipids of the midgut cells and will insert themselves into the membrane, forming β-barrels. Cyt toxin are different in them domains.
Cyt1Aa would facilitate the binding of Cry11Aa to the BBMV receptor, by the interaction of the loop α8, β4 and loop 2 regions of Cry11Aa at the level of the loop β6 – αE and part of β7 of Cyt1Aa. This interaction allows easy insertion of Cry11Aa into membranes.
P20 chaperon and Cry11Aa, Cyt1Aa toxins
Was discovered in B.thuringiensis, a p20 gene in operon with cry11Aa and cyt1Aa. Thus, the P20 protein was shown to be necessary for the formation of inclusion bodies of Cry11Aa and Cyt1Aa. P20 would allow a greater production of these toxins and allow their stability (Cyt1Aa would be protected from the proteolytic action before its maturation). The co-production of P20 and Cyt1Aa in E.coli would limit its toxic effects in the producer cell.
Sequence and Features
- 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]
| None |