Difference between revisions of "Part:BBa K245129"
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| − | <p>Then we identified the protein 3D structure and the ligand it binds to by using I-tasser.</p> | + | <p>Then we identified the protein 3D structure and the ligand it binds to by using I-tasser.Shown in Fig1</p> |
| − | https://2019.igem.org/wiki/images/ | + | |
| − | < | + | <div style="width:image width 300px; font-size:90%; text-align:center;"><img src="https://2019.igem.org/wiki/images/9/92/T--CU--CutA_model1.png" alt="" width="300px" height="300px" ;" /><br>Fig1.Predicted Model with the least free energy</br></div> |
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Revision as of 07:22, 5 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 that predict the protein stability, pI and halophilicity.
| 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 and the ligand it binds to by using I-tasser.Shown in Fig1
Fig1.Predicted Model with the least free energy
Modeling the protein binding to sodium ion with c-score: 0.1
https://2019.igem.org/wiki/images/e/e1/T--CU--2.gifCutA 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. https://2019.igem.org/wiki/images/thumb/2/29/T--CU--cutA1.png/320px-T--CU--cutA1.pngMeasuring 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. https://2019.igem.org/wiki/images/a/a3/T--CU--4.pngWe made replicates from each protein to find the range of concentration produced, we found that using the cell free kit, CSP1,CutA1gave the highest production(figure 2)
Figure 2Testing the binding affinities of the four target proteins.
the binding affinities of the four proteins to Sodium salt was measured using TDS with comparing the affinity of each protein to NaCl, we found that CutA has high affinity to the Sodium salts, lowering the ppm around 900. Figure 3 includes comparison for the four proteins for their ability to reduce sodium chloride. Figure 3We made a comparison between the ability of CutA1 tested protein to reduce TDS of Sodium chloride to the Metal salt that is it originally binds on the other hand, CutA1 has affinity for copper,, so we measured the TDS before and after adding CutA1 to Copper Sulfate solution, We have found that CutA1 reduced the TDS of NaCl solution (fig. 4), correlating well with the results of the protein modelling made in I-tasser.
Sequence and Features