Difference between revisions of "Part:BBa K4765015"

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===Introduction===
 
===Introduction===
 
''Ammopiptanthus nanus'' antifreeze protein (AnAFP) is an antifreeze protein derived from ''Ammopiptanthus nanus''. It protects the stability of membrane structures under cold conditions by binding to or dissociating from the membrane. It prevents the coagulation of macromolecules and plays a regulatory role by binding to or dissociating from calcium ions<ref>Deng, L.-Q., Yu, H.-Q., Liu, Y.-P., Jiao, P.-P., Zhou, S.-F., Zhang,  S.-Z., Li, W.-C., & Fu, F.-L. (2014). Heterologous expression of  antifreeze protein gene AnAFP from ''Ammopiptanthus nanus'' enhances cold  tolerance in ''Escherichia coli'' and tobacco. ''Gene, 539''(1), 132-140.https://doi.org/10.1016/j.gene.2014.01.013</ref>.
 
''Ammopiptanthus nanus'' antifreeze protein (AnAFP) is an antifreeze protein derived from ''Ammopiptanthus nanus''. It protects the stability of membrane structures under cold conditions by binding to or dissociating from the membrane. It prevents the coagulation of macromolecules and plays a regulatory role by binding to or dissociating from calcium ions<ref>Deng, L.-Q., Yu, H.-Q., Liu, Y.-P., Jiao, P.-P., Zhou, S.-F., Zhang,  S.-Z., Li, W.-C., & Fu, F.-L. (2014). Heterologous expression of  antifreeze protein gene AnAFP from ''Ammopiptanthus nanus'' enhances cold  tolerance in ''Escherichia coli'' and tobacco. ''Gene, 539''(1), 132-140.https://doi.org/10.1016/j.gene.2014.01.013</ref>.
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{|
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| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/description/9anti-freeze-module.png" alt="contributed by Fudan iGEM 2023"></html>
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|-
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| '''Figure 1. The principle of antifreeze AnAFP'''
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 +
|}
  
 
===Usage and Biology===
 
===Usage and Biology===
 
We heterologously expressed codon-optimized AnAFP in ''E.coli'', endowing it with antifreeze capability.
 
We heterologously expressed codon-optimized AnAFP in ''E.coli'', endowing it with antifreeze capability.
 
===Characterization===
 
===Characterization===
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====Sequencing map====
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====Sequencing Map====
 
{|
 
{|
 
| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/zsl/anafp-sequence.png" alt="contributed by Fudan iGEM 2023"></html>
 
| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/zsl/anafp-sequence.png" alt="contributed by Fudan iGEM 2023"></html>
 
|-
 
|-
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| '''Figure1 Sequencing map of AnAFP'''
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| '''Figure 2. Sequencing map of AnAFP'''
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Sequencing was performed using the two primers：T7: 5-TAATACGACTCACTATAGG-3 and Kan-F: 5-ATTCTCACCGGATTCAGT-3.
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Sequencing starts from the T7 terminator, with the primer 5-GCTAGTTATTGCTCAGCGG-3.
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|}
 
|}
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AnAFP is accurately expressed in ''E. coli'' BL21 DE3.
 
  
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====AnAFP Antifreeze Capability====
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====Successful Protein Expression====
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{|
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| <html><img style="width:300px" src="https://static.igem.wiki/teams/4765/wiki/zsl/protein-gel/anafp-2.png" alt="contributed by Fudan iGEM 2023"></html>
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|-
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| '''Figure 3. SDS-PAGE electrophoresis of AnAFP'''
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We constructed AnAFP into the pET28a plasmid and transformed it into ''E. coli'' BL21 AI. Lanes 1 to 3 represent AnAFP, AnAFP + ara, and AnAFP + ara + IPTG, as indicated by the red arrow, we successfully expressed AnAFP.
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|}
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 +
====AnAFP Antifreeze Assay ====
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We tested the antifreeze capability of AnAFP and found that ''E. coli'' expressing AnAFP exhibited a higher survival rate under prolonged cold treatment conditions compared to ''E. coli'' transformed with an empty PET28 vector, indicating that it possesses antifreeze ability.
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{|
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| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/zsl/anafp-protocol.png" alt="contributed by Fudan iGEM 2023"></html>
 +
|-
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| '''Figure 4. Workflow of AnAFP Antifreeze Assay'''
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 +
|}
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The experimental group consists of ''E. coli BL21'' DE3 expressing AnAFP, the control group comprises ''E. coli'' BL21 DE3 transformed with the empty vector pET28a. All the groups are  cultured overnight. After reaching an OD600 value of 1.0, they are subjected to cold treatment at 0 °C.Samples are taken at 0, 24, 48, 72 and 96 hours during cold treatment and are diluted 10^5 times in ddH2O for CFU counting. CFU counting is performed with the help of imageJ.
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For each tube of cold-treated ''E. coli'', the colony count at the measurement time is divided by the colony count at 0 hours to obtain the survival rate of ''E. coli'' in that tube at that specific time. The average survival rate for the same type of bacteria is then calculated, and survival curves for each group of ''E. coli'' are plotted.
 
{|
 
{|
 
| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/jzk/measurement-2.png" alt="contributed by Fudan iGEM 2023"></html>
 
| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/jzk/measurement-2.png" alt="contributed by Fudan iGEM 2023"></html>
 
|-
 
|-
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| '''Figure2 Colonies at 0,24,48,72 and 96 hours of cold treatment at 0 °C'''
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| '''Figure 5. Colonies at 0,24,48,72 and 96 hours of cold treatment at 0 °C'''
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The experimental group consists of ''E. coli BL21'' DE3 expressing AnAFP, the control group comprises ''E. coli'' BL21 DE3 transformed with the empty vector PET28. All the groups are  cultured overnight. After reaching an OD value of 1.0, they are subjected to cold treatment at 0 °C.Samples are taken at 0, 24, 48, 72 and 96 hours during cold treatment and are diluted 10^5 times in ddH2O for CFU counting. CFU counting is performed with the help of imageJ.
+
From left to right is a time gradient of freezing treatment, while from up to down are the groups that were transformed with AnAFP and the control group.
 
|}
 
|}
  
 
{|
 
{|
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| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/zsl/anafp-survival-curve.png" alt="contributed by Fudan iGEM 2023"></html>
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| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/zsl/anafp-survival-curve1.png" alt="contributed by Fudan iGEM 2023"></html>
 
|-
 
|-
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| '''Figure3 Survival curves under cold treatment at 0 °C'''
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| '''Figure 6. Survival curves under cold treatment at 0 °C'''
−
For each tube of cold-treated ''E. coli'', the colony count at the measurement time is divided by the colony count at 0 hours to obtain the survival rate of ''E. coli'' in that tube at that specific time. The average survival rate for the same type of bacteria is then calculated, and survival curves for each group of ''E. coli'' were plotted.
+
In the initial 48 hours, there was no significant difference in the survival rates between the experimental and control groups. However, after 48 hours, the survival rate of the experimental group was significantly higher than that of the control group.
 
|}
 
|}
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In the initial 48 hours, there was no significant difference in the survival rates between the experimental and control groups. However, after 48 hours, the survival rate of the experimental group was significantly higher than that of the control group. This indicates that AnAFP possesses the ability to allow ''E. coli'' to survive for an extended period in cold environments.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 15:22, 12 October 2023


AnAFP contributed by Fudan iGEM 2023

Introduction

Ammopiptanthus nanus antifreeze protein (AnAFP) is an antifreeze protein derived from Ammopiptanthus nanus. It protects the stability of membrane structures under cold conditions by binding to or dissociating from the membrane. It prevents the coagulation of macromolecules and plays a regulatory role by binding to or dissociating from calcium ions[1].

contributed by Fudan iGEM 2023
Figure 1. The principle of antifreeze AnAFP

Usage and Biology

We heterologously expressed codon-optimized AnAFP in E.coli, endowing it with antifreeze capability.

Characterization

Sequencing Map

contributed by Fudan iGEM 2023
Figure 2. Sequencing map of AnAFP

Sequencing starts from the T7 terminator, with the primer 5-GCTAGTTATTGCTCAGCGG-3.

Successful Protein Expression

contributed by Fudan iGEM 2023
Figure 3. SDS-PAGE electrophoresis of AnAFP

We constructed AnAFP into the pET28a plasmid and transformed it into E. coli BL21 AI. Lanes 1 to 3 represent AnAFP, AnAFP + ara, and AnAFP + ara + IPTG, as indicated by the red arrow, we successfully expressed AnAFP.

AnAFP Antifreeze Assay

We tested the antifreeze capability of AnAFP and found that E. coli expressing AnAFP exhibited a higher survival rate under prolonged cold treatment conditions compared to E. coli transformed with an empty PET28 vector, indicating that it possesses antifreeze ability.

contributed by Fudan iGEM 2023
Figure 4. Workflow of AnAFP Antifreeze Assay

The experimental group consists of E. coli BL21 DE3 expressing AnAFP, the control group comprises E. coli BL21 DE3 transformed with the empty vector pET28a. All the groups are cultured overnight. After reaching an OD600 value of 1.0, they are subjected to cold treatment at 0 °C.Samples are taken at 0, 24, 48, 72 and 96 hours during cold treatment and are diluted 10^5 times in ddH2O for CFU counting. CFU counting is performed with the help of imageJ.

For each tube of cold-treated E. coli, the colony count at the measurement time is divided by the colony count at 0 hours to obtain the survival rate of E. coli in that tube at that specific time. The average survival rate for the same type of bacteria is then calculated, and survival curves for each group of E. coli are plotted.

contributed by Fudan iGEM 2023
Figure 5. Colonies at 0,24,48,72 and 96 hours of cold treatment at 0 °C

From left to right is a time gradient of freezing treatment, while from up to down are the groups that were transformed with AnAFP and the control group.

contributed by Fudan iGEM 2023
Figure 6. Survival curves under cold treatment at 0 °C

In the initial 48 hours, there was no significant difference in the survival rates between the experimental and control groups. However, after 48 hours, the survival rate of the experimental group was significantly higher than that of the control group.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 451


References

  1. ↑ Deng, L.-Q., Yu, H.-Q., Liu, Y.-P., Jiao, P.-P., Zhou, S.-F., Zhang, S.-Z., Li, W.-C., & Fu, F.-L. (2014). Heterologous expression of antifreeze protein gene AnAFP from Ammopiptanthus nanus enhances cold tolerance in Escherichia coli and tobacco. Gene, 539(1), 132-140.https://doi.org/10.1016/j.gene.2014.01.013