yeast culture is diluted before measuring OD (1ml overnight culture in 4 ml SDA media ) The OD600 was found to vary with the different concentrations, suggesting that the optimum NaCl concentration for the yeast growth was 50g/L
Before Adding the yeast Sodium concentration was measured and equals = 2740 ppm By the addition of yeast sodium chloride concentration began to differ along time intervals, at around 1hr the yeast began to release the salt back into the media.
The first experiment performed for the production of CutA1, CSP1, CRS5, Bacterioferritin was testing the best time of production using the calculated theoretical Extinction coefficient found in the following table for each protein:
The protein was produced using Ls70 linear expression cell-free kit, under the control of constitutive family member promotor "BBa_J23102” and strong RBS “BBa_B0034” ● We identified protein concentration by measuring the absorbance at 280nm, concentration is calculated by beer’s law(A = εbc) using the identified theoretical extinction coefficient[table1]. ● We plotted the time of reaction(x-axis) vs Concentration of the protein produced(y-axis).[Fig.1]
● We made replicates from each protein to find the range of concentration produced, we found that using the cell free kit; CSP1,CutA1 gave the highest production.[Fig.2]
● The binding affinities of the four proteins to Sodium salt was measured using TDS by comparing the affinity of each protein to NaCl. -It was found that CutA1 has the highest affinity to the Sodium salts, lowering the ppm around 900. In case of CRS5 and Bacterioferritin -in contrast to CutA- the TDS decreased by 300, 330 ppm. For the case of CSP1, the TDS of the NaCl after adding the protein was the least 70 ppm.[Fig.3]
● We made a comparison between the ability of the tested proteins to reduce TDS of Sodium chloride and other metal salt that it originally binds to,both CutA1 and CSP1 have affinity for copper, while bacterioferritin has affinity to bind ferric, so we measured the TDS before and after adding CSP1,CutA1 to Copper Sulfate solution, and the TDS before and after adding bacterioferritin to ferric Chloride Solutions. We have found that CutA1 reduced the TDS of NaCl solution [fig. 4], correlating well with the results of the protein modelling made using I-tasser,while CSP1 has been found to possess higher affinity to sequester copper than sodium salt from the solution [fig.5].Also the bacterioferritin seemed to have higher affinity for the ferric than sodium salts[fig.6]
● As for GST+CRS5 protein, the protein reduced the amount of TDS in NaCl and CuSO4 Solutions; with reduction in TDS 288,390 ppm, respectively[Fig. 7]
● In order to know the best and least temperature at which the proteins bind to the metals, we performed the experiment at different temperatures 4,25,37,42,68 Celsius. The results showed that: CSP1 best temperature to reduce the TDS of NaCl solution is 4 degree with reduction reaching 1300 ppm, for CutA protein best worked in reduction of TDS at 37 degree reducing TDS around 600 ppm; on the other hand Bacterioferritin didn’t give significant results at room temperature, but worked its best at 42 degree in reducing total dissolved salts [Fig. 8]
● While in case of applying solution of the same concentration of NaCl adjusted to different pHs ranged from 3-9 to CSP1,Bacterioferritin, both of them highly reduced the value of TDS to around 1200, 1506 ppm respectively. Showing better reduction and binding in acidic media than in Basic media (Fig. 9)
● We used tac promoter instead of a constitutive promoter, as we were working on Gst tagged protein,so we want to ensure the right folding for the protein and avoid clustering and crowding, we have chosen tac promoter to control Bacterioferritin-Gst tagged protein, with adding different concentrations of IPTG to induce the transcription of the downstream gene, when we induced cell-free expression, we found that the best concentration of IPTG could be added for induction in Sigma70 expression kit is 1mM IPTG (Fig. 10)
● According to the modeling we performed, we combined GST and Bacterioferritin in one plasmid under the expression of tac promoter considering the folding time of the fused protein, then we tested the reduction of the TDS value of NaCl solution at room temperature, because bacterioferritin didn’t reduce TDS at room temperature unlike in 42 degree.From the results we found a great improvement in the ability of the tagged bacterioferritin to reduce the TDS of NaCl solution at room temperature, ~5 folds higher TDS reduction, enabling the fusion protein to behave better in further application of the system (Fig.11)
