Team:Athens/Proof Of Concept

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iGEM_Athens_2021_AdAPTED

Proof of Concept

As stated in the Proposed Implementation, AdAPTED can be used for the acquisition of the two reagents, dNTPs and Pfu polymerase, in three different forms. Those are as lyophilized recombinant plasmid, as lyophilized cell lysate and as isolated and purified reagents. All three ideas can be implemented for the production of dNTPs, whereas Pfu polymerase can only be produced from the lyophilized plasmid or purified.

Here, we present a proof of concept of the final products that will be distributed from this project.

The plasmid pPfuT7 was successfully ligated and used for the transformation of E. coli BL21 (DE3) cells through Golden Gate Assembly, as it is described in Protocols. The transformed cells can be seen in Figure 1.

Figure 1: Transformed E. coli cells

The recombinant plasmid containing Pfu polymerase was isolated from the transformed bacteria using the Monarch Plasmid Miniprep Kit. The plasmid was digested with the PstI restriction enzyme and electrophoresed in both digested and undigested forms, as seen in Figure 2. The plasmid pPfuT7 has only one restriction site for PstI and its size is close to 4.5 kb. In this way, we confirmed that the ligation and transformation were successful. Thus the isolated plasmid is ready to be amplified through PCR, lyophilized and distributed to the users.

Figure 2: pPfuT7 electrophoresis: (left) undigested supercoiled plasmid and (right) digested pPfuT7 with PstI.

To do that, we first had to assess if the bacteria could actually produce Pfu polymerase. His-tag purification was executed from three of the colonies that were obtained from transformation, in order to test the repeatability of the method. After the purification, SDS-PAGE was performed. The 10 mM and 100 mM imidazole elution solutions for all three cultures were loaded in SDS-PAGE, as shown in Figure 3. For culture 2, 5 mM (not shown in Figure 3) and the second 100 mM imidazole elution solutions were also loaded. The polymerase was expected to be eluted in imidazole solution and especially in the three 100 mM elutions. However it is seen that the protein is found at the 10 mM imidazole solution. That can be attributed to the fact that Pfu polymerase is a large protein (92 kDa) and it is easier to leave the column. This method proved that the elution solutions contained an isolated protein with molecular weight close to 93 kDa which matches the molecular weight of Pfu polymerase.

Figure 3: SDS-PAGE of Pfu solution post chromatography. From left to right: isolated proteins derived from cell cultures 2, 2’ and 3 that are eluted in imidazole solutions of different concentrations; 10 mM and 100mM.

In order to measure the concentration of Pfu polymerase, spectrophotometry of all consecutive elution solutions, as described in Protocols, was performed. The results of the spectrophotometry are shown in Table 1. The control solution of imidazole was measured at 0.294.

Table 1: Photometry measurements (the control has been subtracted)
Culture number 3 2’ 2
10 ml Talon 3.049 2.730 2.830
7 ml imid 5 mM 0.666 0.695 0.735
7 ml imid 10 mM 0.258 0.268 0.364
4 ml imid 100 mM 0.067 0.048 0.127
6 ml imid 100 mM 0.047 0.044 0.188
6 ml imid 100 mM 0.017 0.019 0.065
Flowthrough 2.926 3.158 2.977

To calculate the concentration of the protein, the Lambert-Beer law is used with cuvette length b=0.67 cm and proportionality constant ε=129290. The results for the final concentration of Pfu polymerase in all five imidazole solutions in g/L are calculated are shown in Table 2. The SDS-PAGE method showed that Pfu polymerase was isolated in the 10 mM imidazole elution solution. So the average value of the spectrophotometry measurement that was taken from this solution for all three colonies corresponds to 0.0022 g or 2.2 mg of Pfu in these 7 mL. These measurements were the result of a 500 mL liquid culture, so it can be said that the system produces on average 4.4 mg of Pfu per Liter of liquid culture.

Table 2: Pfu polymerase concentration in imidazole elution solution
Culture number 3 2’ 2
10 ml Talon 3.049 2.730 2.830
7 ml imid 5 mM 0.666 0.695 0.735
7 ml imid 10 mM 0.258 0.268 0.364
4 ml imid 100 mM 0.067 0.048 0.127
6 ml imid 100 mM 0.047 0.044 0.188
6 ml imid 100 mM 0.017 0.019 0.065
Flowthrough 2.926 3.158 2.977

In conclusion, we were able to create the pPfuT7 plasmid, successfully use it for transformation and confirm that the recombinant bacteria produce Pfu polymerase. The same could not be done for the dNTPs since technical issues and time limitations hindered us from achieving the ligation of the vector. Nevertheless, the design of the proposed system is complete and the protocols for the dNTP purification and measurement are already designed.