Difference between revisions of "Part:BBa K245129"
| Line 89: | Line 89: | ||
| − | |||
| − | |||
| + | <h1><b>Testing the binding affinities of CutA1.</h1></b> | ||
| + | We made a comparison between the ability of CutA1 protein to reduce both the TDS of Sodium salt and the Metal salt that it originally binds to; which is in this case Copper. So we measured the TDS before and after adding CutA1 to Copper Sulfate solution and Sodium Chloride Solution , We have found that CutA1 reduced the TDS of NaCl solution by approximately 900ppm [fig. 4], correlating well with the results of the protein modelling made in I-tasser. | ||
| − | + | <img src=" https://2019.igem.org/wiki/images/thumb/d/d6/T--CU--tds1.png/800px-T--CU--tds1.png" alt"" width="500px" height="300px" ;" /> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <img src=" https://2019.igem.org/wiki/images/thumb/d/d6/T--CU--tds1.png/800px-T--CU--tds1.png" alt"" width=" | + | |
| − | + | ||
| + | <br> | ||
| + | <br> | ||
<!-- --> | <!-- --> | ||
Latest revision as of 19:41, 12 December 2019
CutA1
This part is the coding sequence for a trimerization domain - CutA1.
Usage and Biology
Characterisation by CU 2019 team
In silico protein modeling
First step in characterising CutA1(BBa_k245129), we used the Expasy ProtParam tool, where the theoretical protein extinction coefficient has been identified in addition to several other factors[Table1].
| Extiction Coeficcient | Gravy | Molecular Weight | PI | Instability Index | Aliphatic Index | kDa |
|---|---|---|---|---|---|---|
| 20690 | -0.037 | 17791.29 | 5.40 | 40.03 "unstable" | 85.84 | 12.321 |
Then we identified the protein 3D structure[fig.1] and the ligand it binds to[fig.2] using I-tasser. I-tasser was used to view the folding structure and predict its affinity to bind metal ligands in addition, to confirm the molecular function. where the model generated was closely related to Crystal structure of possible CutA1 divalent ion tolerance protein from Cryptosporidium parvum Iowa II, with TM-Score 0.95, and sequence coverage 0.9991.
Fig1.Predicted Model with the least free energy
Fig.2 Model showing protein binding to sodium ion with c-score: 0.1
The biological process identified in the model in response to metal ion, through the ligand binding site prediction,predicts that CutA1 could bind to sodium ion;Gene ontology predicted in the model belonged to copper binding.
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 was calculated by beer’s law (A = εbc) using the theoretical extinction coefficient shown in Table1. We then plotted the time of reaction vs the concentration of the protein produced. It was found that the highest production was after 14hrs[fig.3]
Fig3.Showing CutA1 conc at different time intervals
Testing the binding affinities of CutA1.
We made a comparison between the ability of CutA1 protein to reduce both the TDS of Sodium salt and the Metal salt that it originally binds to; which is in this case Copper. So we measured the TDS before and after adding CutA1 to Copper Sulfate solution and Sodium Chloride Solution , We have found that CutA1 reduced the TDS of NaCl solution by approximately 900ppm [fig. 4], correlating well with the results of the protein modelling made in I-tasser.
Sequence and Features