Revision as of 02:58, 30 September 2024

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

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 982
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 982
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 1087
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 982
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 982
Illegal AgeI site found at 157
1000
COMPATIBLE 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.

Figure 1. Cry6Aa2-6*His(pET28a)plasmid vector

By adding His tag to the end of Cry6Aa2, we constructed an expression and purification vector of Cry6Aa2 on plasmid pET28a and transformed the constructed vector into Escherichia coli BL21(DE3).

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

Five of the six colonies were successfully constructed, and the colonies were sequenced after expanded culture. Colony 1 and colony 2 were correct.

Cry6Aa2 expression condition

The bacteria were inoculated in 15 mL LB medium of 50 μg/ml kanamycin sulfate and cultured at 37℃ for 8 h. 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 8000 rpm 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 12000 rpm at 4℃ for 15 min, 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. 5 ml 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 280 nm. Eluting with twice the column volume of the Elution Buffer, 2 ml eluent is collected at a time until the absorbance of the eluent approaches the baseline at 280 nm. 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.

@@ Line 27: / Line 27: @@
 = 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.
+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 H<sub>2</sub>O and 1 μL colony.
 == Agarose gel electrophoresis ==
 The success of vector construction was verified by colony PCR of <i>Escherichia coli</i> BL21(DE3)
@@ Line 41: / Line 41: @@
 == Cry6Aa2 expression condition ==
-The bacteria were inoculated in 15ml LB medium of 50 μg/ml kanamycin sulfate and cultured at 37℃ for 8h. When OD<sub>600</sub> 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.
+The bacteria were inoculated in 15 mL LB medium of 50 μg/ml kanamycin sulfate and cultured at 37℃ for 8 h. When OD<sub>600</sub> 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 cultured 15ml bacterial solution was centrifuged at 8000 rpm 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.
 <br>
-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.
+The cell lysate was obtained by ultrasonic crushing of lytic bacteria, centrifuged at 12000 rpm at 4℃ for 15 min, and the supernatant was transferred to a new EP tube.
 <br>
-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.
+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. 5 ml 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 280 nm. Eluting with twice the column volume of the Elution Buffer, 2 ml eluent is collected at a time until the absorbance of the eluent approaches the baseline at 280 nm. The purified sample was obtained.
 <br>
 == SDS-PAGE ==
@@ Line 59: / Line 59: @@
 </html>
 <br>
-Bacterial protein SDS-PAGE and purified bacterial protein SDS-PAGE demonstrated the successful expression of Cry6Aa2 in <i>Escherichia coli</i> 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.
+Bacterial protein SDS-PAGE and purified bacterial protein SDS-PAGE demonstrated the successful expression of Cry6Aa2 in <i>Escherichia coli</i> BL21(DE3).Cry6Aa2 protein with 6*His has a molecular weight of 49 kDa.In SDS-PAGE for Bacterial protein,compared with the Ctrl, the samples all had obvious bands at 49 kDa.In SDS-PAGE for purification of bacterial protein, bands were visible in the induction group at 49 kDa.
 == Western Blot ==
 <html>
@@ Line 65: / Line 65: @@
      <img src="https://static.igem.wiki/teams/5335/cry6aa2/wb.png" style="width:70%; ">
 <br><b>Figure 4.  </b></center>Western Blot of Cry6Aa2-6*His.M:Protein marker;1:Uninduced group2:Induction group</br>
-ul 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.
+ul 5x protein loading buffer was added to the 80μL purified sample, the sample was prepared at 98℃ for 15 min, the sample was added, electrophoresis was performed at 80 v for 2 h, Transfer to nitrocellulosic membrane(It has been activated by methanol) on ice at 14 v, 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.
      </div>
 </div>
 </html>
 <br>
-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 <i>Escherichia coli</i> BL21(DE3), or by low-dose leakage of lacⅠ operons.
+Western Blot further demonstrated the successful expression of Cry6Aa2. There were coarse bands at 49 kDa 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 49 kDa might be caused by other proteins in <i>Escherichia coli</i> BL21(DE3), or by low-dose leakage of lacⅠ operons.
 == Reference ==
 .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.
 <br>
 .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.

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