Difference between revisions of "Part:BBa K4765015"

Revision as of 04:10, 10 October 2023

AnAFP contributed by Fudan iGEM 2023

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

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

Figure 2. Colonies at 0,24,48,72 and 96 hours of cold treatment at 0 °C

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.

Figure 3. 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. This indicates that AnAFP possesses the ability to allow E. coli to survive for an extended period in cold environments. 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

↑ 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

[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

[1]

@@ Line 24: / Line 24: @@
 | <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/jzk/measurement-2.png" alt="contributed by Fudan iGEM 2023"></html>
 |-
-| '''Figure2 Colonies at 0,24,48,72 and 96 hours of cold treatment at 0 °C'''
+| '''Figure 2. Colonies at 0,24,48,72 and 96 hours of cold treatment at 0 °C'''
 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.
 |}
@@ Line 31: / Line 31: @@
 | <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>
 |-
-| '''Figure3 Survival curves under cold treatment at 0 °C'''
+| '''Figure 3. 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.
 |}