Part:BBa_K245129

CutA1

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

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

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

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.

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. https://2019.igem.org/wiki/images/a/a3/T--CU--4.png

We 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 2

Testing 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 3

We 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 BBa_K245129 SequenceAndFeatures