Difference between revisions of "Part:BBa K5335006"
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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 kDa.Cry6Aa2 can be expressed in E. coli BL21 (DE3) and exert its toxicity.
 
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 kDa.Cry6Aa2 can be expressed in E. coli BL21 (DE3) and exert its toxicity.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5335006 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K5335006 parameters</partinfo>
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===Usage and Biology===
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= Usage and Biology =
 
= Usage and Biology =
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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 pathway. 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.
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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 pathway. 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.
 
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<center><img src="https://static.igem.wiki/teams/5335/cry6aa2/cry.png" style="width:40%; "></center>
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<center><img src="https://static.igem.wiki/teams/5335/cry6aa2/huilutu.png" style="width:40%; "></center>
 
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<center><b>Figure  </b></center>
 
<center><b>Figure  </b></center>
 
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).
 
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).
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= Characterization =
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== Colony PCR ==
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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.
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== Agarose gel electrophoresis ==
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The success of vector construction was verified by colony PCR of <i>Escherichia coli</i> BL21(DE3)
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    <img src="https://static.igem.wiki/teams/5335/cry6aa2/clony-pcr.png" style="width:55%; ">
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<br><b>Figure  </b></center>M: DL2000 DNA Marker; 1-6: Colony PCR bands for <i>Escherichia coli</i> Top 6</br>
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Five of the six colonies were successfully constructed, and the colonies were sequenced after expanded culture. Colony 1 and colony 2 were  correct.
  
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== Cry6Aa2 expression condition ==
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===Functional Parameters===
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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.
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<partinfo>BBa_K5335006 parameters</partinfo>
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== Purification of Cry6Aa2 ==
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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.
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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.
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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.
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== SDS-PAGE ==
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    <img src="https://static.igem.wiki/teams/5335/cry6aa2/sds-page.png" style="width:70%; ">

Revision as of 07:42, 29 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 kDa.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 pathway. 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. What？


Figure
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 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 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