Part:BBa_K5335006
Cry6Aa2 is a Nematocidal crystal protein from Bacillus thuringiensis YBT-1518
Cry6Aa2 is a pesticidal crystal protein that mainly in the gut of nematodes by triggering a specific necrosis pathway different from other Cry proteins.Total structure weight of Cry6Aa2 protein is 48.23 kDa1.Cry6Aa2 can be expressed in E. coli BL21 (DE3) and exert its toxicity.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 982
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 982
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1087
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 982
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 982
Illegal AgeI site found at 157 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
As a Gram-positive bacterium, Bacillus thuringiensis produces a variety of insecticidal crystal proteins during its growth. In the past hundred years, Bacillus thuringiensis has been widely developed and studied as a microbial insecticidal agent. The gene encoding the insecticidal crystal protein is the main source of transgenic insect-resistant plants. Cry6Aa2 is a crystal protein from Bt YBT-1518 that kills nematodes through a specific necrotic pathway2. In our project, we fused Cry6Aa2 with SpyCatcher, attached it to virus-like particles with SpyTag, and released it into the soil environment to kill nematodes through the cleavage of engineered bacteria.
Characterization
Colony PCR
We designed an forward primer inside the Cry6Aa2 gene sequence and a reverse primer on a linearized vector to serve as a primer for our colony PCR, as this may reduce the production of false positives.We chose 2× Magic Green Taq SuperMix.It contains DNA Polymerase, dNTP, and an loading buffer system.The PCR reaction consisted of 15 μL 2 × Magic Green Taq SuperMix, 1.5 μL forward primer (3.75 mM), 1.5 μL reverse primer (3.75 mM), 11 μL H2O and 1 μL colony.
Agarose gel electrophoresis
The success of vector construction was verified by colony PCR of Escherichia coli BL21(DE3)
Figure 2. M: DL2000 DNA Marker; 1-6: Colony PCR bands for Escherichia coli Top 6
Cry6Aa2 expression condition
The bacteria were inoculated in 15ml LB medium of 50 μg/ml kanamycin sulfate and cultured at 37℃ for 8h. When OD600 reached 1.0, the induction group was induced with 1M IPTG at 16℃ overnight. The non-induced group was cultured overnight at 16℃ without IPTG.
Purification of Cry6Aa2
The cultured 15ml bacterial solution was centrifuged at 8000rpm at 4℃ for 10min to discard the supernatant. An appropriate amount of PBS was added to re-suspend the washing bacteria, and then the previous centrifugal operation was repeated. The bacterial suspension was obtained by re-suspension with 3ml Binding Buffer.
The cell lysate was obtained by ultrasonic crushing of lytic bacteria, centrifuged at 12000rpm at 4℃ for 15min, and the supernatant was transferred to a new EP tube.
Cry6Aa2 protein was purified by Ni column using gravity method. The supernatant and Binding Buffer were mixed in equal amounts to make the sample, and the sealing solution was slowly discharged. 5ml Washing Buffer was used to balance the Ni column, and the sample was placed on the column at twice the column volume each time, and the first washing solution was placed on the column again.Wash the column with twice the column volume of Washing Buffer and collect the runthrough until the runthrough absorbance approaches the baseline at 280nm. Eluting with twice the column volume of the Elution Buffer, 2ml eluent is collected at a time until the absorbance of the eluent approaches the baseline at 280nm. The purified sample was obtained.
SDS-PAGE
Figure 3. A:SDS-PAGE for Bacterial protein;B: SDS-PAGE for purification of bacterial protein M:Protein marker;Ctrl:Escherichia coli BL21(DE3)without an imported vector.1, 2, 3 and 4 were Escherichia coli BL21(DE3) introduced into Cry6Aa2-6*His(pET28a).Among them, 1 and 3 were induced without IPTG, and 2 and 4 were induced by 1 M IPTG.
Bacterial protein SDS-PAGE and purified bacterial protein SDS-PAGE demonstrated the successful expression of Cry6Aa2 in Escherichia coli BL21(DE3).Cry6Aa2 protein with 6*His has a molecular weight of 49kDa.In SDS-PAGE for Bacterial protein,compared with the Ctrl, the samples all had obvious bands at 49kDa.In SDS-PAGE for purification of bacterial protein, bands were visible in the induction group at 49kDa.
Western Blot
Figure 4. Western Blot of Cry6Aa2-6*His.M:Protein marker;1:Uninduced group2:Induction group 20ul 5x protein loading buffer was added to the 80ul purified sample, the sample was prepared at 98℃ for 15min, the sample was added, electrophoresis was performed at 80v for 2h, Transfer to nitrocellulosic membrane(It has been activated by methanol) on ice at 14v, the primary antibody(His-Tag Mouse Monoclonal Antibody) was incubated, eluted, the secondary antibody(Goat anti-Mouse IgG) was incubated, and the color was eluted.
Western Blot further demonstrated the successful expression of Cry6Aa2. There were coarse bands at 49kDa in the induced group, and the protein content was significantly different from that in the non-induced group.We hypothesized that the bands in the uninduced group near 49kDa might be caused by other proteins in Escherichia coli BL21(DE3), or by low-dose leakage of lacⅠ operons.
Reference
1.Zhang F, Peng D, Cheng C, Zhou W, Ju S, Wan D, Yu Z, Shi J, Deng Y, Wang F, Ye X, Hu Z, Lin J, Ruan L, Sun M. Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1). PLoS Pathog. 2016 Jan 21;12(1):e1005389. doi: 10.1371/journal.ppat.1005389.
2.Wang P, Zhang C, Guo M, Guo S, Zhu Y, Zheng J, Zhu L, Ruan L, Peng D, Sun M. Complete genome sequence of Bacillus thuringiensis YBT-1518, a typical strain with high toxicity to nematodes. J Biotechnol. 2014 Feb 10;171:1-2. doi: 10.1016/j.jbiotec.2013.11.023. Epub 2013 Dec 11.