Coding
RFC 37

Part:BBa_K245129

Designed by: Špela Miklavič   Group: iGEM09_Slovenia   (2009-10-16)
Revision as of 03:13, 20 October 2019 by Lina (Talk | contribs)

CutA1

This part is the coding sequence for a trimerization domain - CutA1.

Usage and Biology

In silico protein modeling

Using Expasy ProtParam tool*, theoretical protein extinction coefficient has been identified in addition to several other factors that predict the protein stability, pI and halophilicity.

T--CU--Table1.png

After which we aimed to identify the protein 3D structure and which ligand it binds by using molecular dynamics protein function prediction tool which is I-tasser. Id of the work submitted is: S494196

Predicted Model with the least free energy

T--CU--1.gif


The biological process identified in the model in response to metal ion, through the ligand binding site prediction, we were able to predict that CutA could bind to sodium ion;Gene ontology predicted in the model belonged to copper binding.

Modeling the protein binding to sodium ion with c-score: 0.1

T--CU--2.gif

CutA production using Linear expression cell-free kit

The protein produced using Ls70 linear expression cell-free kit, under the control of constitutive family member promotor (J23102 ) and strong RBS ( B0034). We identified protein concentration by measuring the absorbance at 280nm, concentration is calculated by beer’s law using the previously identified theoretical extinction coefficient. We plotted the time of reaction vs the concentration of the protein produced to characterize the part production using the linear cell-free kit.

320px-T--CU--cutA1.png


Measuring cutA Binding Affinity

We identified the best production time which is 14 hours, then we expressed the protein to test its affinity of binding to copper and sodium based on the modeling results, using TDS meter,we measured the decrease of 159g/l and 70g/l copper sulfate and sodium chloride,respectively. We made replicates and took the average, CutA was able to decrease NaCl by 2 parts per million. However, it has a much higher affinity to copper where it reduced the amount of Copper sulfate by 0.3 part per thousand.


T--CU--4.png

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
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None