As this year’s iGEM team at RWTH Aachen, we achieved our goal of designing a modified phosphate binding protein (PBP) for phosphate recycling. The aim of this modification is to overcome the harsh external conditions such as high pH, which are normally needed to release phosphate bound in PBPs in vitro . Therefore, we have created a fusion construct of a PBP (2ABH) and a photosensitizer, namely SOPP3. To investigate the phosphate recycling capabilities of the protein construct SOPP3-2ABH, we had to transform the designed gene into a production organism. After successful transformation, we needed to find working conditions for the expression of the gene, as well as for its purification and immobilization. These aspects are covered in the engineering success section. Proving our concept, we needed to determine whether our construct was still able to bind phosphate and could release the phosphate after exposure to light.
Determination of Phosphate Binding
After we managed to immobilize our protein construct, the next step was to test its phosphate binding capabilities. Therefore, we added a phosphate solution with known concentration to our immobilized 2ABH-SOPP3. After a short incubation time, samples were taken to measure the concentration of phosphate left in the supernatant (see Experiments). The samples were analyzed using the Phosfinity Assay and the results showed a declining phosphate concentration in the supernatant. For more information see our results section. In order to make the results comparable, the same procedure was done with non-modified 2ABH. This approach showed a decrease of phosphate in the supernatant as well. Due to our experimental design, a decrease of phosphate in the supernatant indicates successful binding of phosphate to the PBP. Considering the successful immobilization of the SOPP3-2ABH and the decrease of phosphate, we proved that the phosphate binding capabilities of 2ABH are contained, and the photosensitizer has no negative impact on it.
Effect of Illumination
To identify the effect of illumination with blue light on our PBPs, we decided to investigate its impact on the sole phosphate binding protein 2ABH first. The aim of this experiment was to show that illumination with blue light alone has no effect on the phosphate binding ability of 2ABH. We illuminated the non-modified 2ABH, after phosphate binding was done. Samples were taken after specific illumination times (see Experiments) and the phosphate concentration was measured using the Phosfinity Assay. The experiments showed no significant increase of phosphate in the supernatant following the illumination with blue light (see Results). This proves that blue light illumination alone has no impact on the binding capabilities of 2ABH.
As the impact of blue light on the PBP alone has been excluded, we aimed to analyze its impact on the construct containing the photosensitizer SOPP3. We carried out the same experiment for the SOPP3-2ABH construct as for lone 2ABH. To this end, we exposed the immobilized construct with already bound phosphate to blue light. Samples were taken after the same illumination times and were similarly analyzed. The results showed an increase of phosphate in the supernatant for the samples taken up to 30 minutes. After 30 minutes of illumination, no significant increase of phosphate could be detected (see Results). This proves two points. First, the photosensitizer had the desired effect. Upon illumination, reactive oxygen species (ROS) have been produced, leading to the deconstruction of the 2ABH. Therefore, the phosphate binding protein loses its function, and the phosphate is set free. Second, the fast effect of SOPP3 is shown. After 30 minutes of illumination, SOPP3 produced enough ROS to cause the release of almost the entire amount of bound phosphate. Longer illumination times are not needed for complete release.
As statistic reliability of experimental results is an important part of science, we decided to perform statistic testing to evaluate the illuminations results. For this purpose, we used Student's t-test with two independent samples. At first, we tested the statistic resilience of the phosphate binding of SOPP3-2ABH. We performed a Student's t-test, where one sample was the phosphate concentration of the stock solution and the other was the phosphate concentration in the supernatant after phosphate binding to the immobilized SOPP3-2ABH. In addition, we tested the statistic resilience of the phosphate release. For that reason, we compared the phosphate concentration in the supernatant with the phosphate concentration of the step before (10 minutes of illumination is compared to 0 minutes of illumination, 30 minutes to 10 minutes and 60 minutes to 30 minutes), using the Student's t-test.
We chose a significance level of 95%. The sample sizes were under 25 and we assumed normal distribution as well as variance similarity.
Table 1: Measured test variables of Student's t-test for the comparison of the samples taken at consecutive time stamps. The value for the significance level of 5% was chosen as the significance level of the sample comparison with the smallest degree of freedom (df=8), which is consequently the highest absolute value to reach in our Student's t-test for statistic approval.
|Stock to 0 minutes of illumination
|0 to 10 (minutes of illumination)
|10 to 30 (minutes of illumination)
|30 to 60 (minutes of illumination)
|Test variable t (absolute value)
|value for significance level 95%
Because the absolute value of the test statistic t is greater than the significance level value (table 1), Student's t-test shows that the release of phosphate from SOPP3-2ABH at 10 minutes and 30 minutes is not random but induced by blue light illumination with a probability of 95%. After 60 minutes of illumination, the change in phosphate concentration of the supernatant compared to that after 30 minutes of illumination is indistinguishable from random fluctuations or measurement errors. This is indicated by the absolute value of the test statistic t being less than the value required for the 95% significance level (table 1). The original binding of phosphate to SOPP3-2ABH is no coincidence as well, as the absolute value of the test statistic t is greater than the required value for the 95% significance level (table 1).
It has been demonstrated that our modified PBP not only functions as intended in terms of phosphate binding but is also capable of releasing phosphate under blue light irradiation. Thus, we have achieved our goal of overcoming the difficult conditions of phosphate release from PBPs. To optimize this phosphate recycling concept, the next goal should be the implementation of a reversible process. On this given proof-of-concept, we already worked with a light-switchable protein, VVD (see Results).