Part:BBa_K4765015
AnAFP
Contents
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].
Usage and Biology
We heterologously expressed codon-optimized AnAFP in E.coli, endowing it with antifreeze capability.
Characterization
Sequencing map
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| Figure 1. Sequencing map of AnAFP
Sequencing is performed using the primer：5-GCTAGTTATTGCTCAGCGG-3 |
AnAFP is accurately expressed in E. coli BL21 DE3.
AnAFP Antifreeze Capability
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.
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.
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.
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| Figure 2. 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. |
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| Figure 3. 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. |
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. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 451
References
- ↑ 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
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