Team:Athens/Implementation
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Proposed Implementation
Introduction
The project AdAPTED is a unique approach for the production of dNTPs and Pfu polymerase, providing an ecological and affordable alternative for two much needed reagents. Along with pieces of information that were gathered throughout the year from interviews with experts and from the survey answers we got from researchers and doctors who use Nucleic Acid Amplification Techniques (NAAT), that are all described in the Human Practices page in detail, we shaped the alternatives for the final forms of the end product. Because our vision for this project was to make an impact and contribute to the communities that frequently perform NAAT and need the reagents, we did not want to restrict or limit the implementation of the project. NAAT techniques include PCR and LAMP, two very common methods used daily around the world for different applications. Communities who perform NAAT range from researchers who use them as part of their experimental procedure, doctors and diagnostic laboratories professionals who use them to diagnose patients in hospitals or at the field, to Biology related fields students and high school teachers who use them for educational purposes.
After defining the aim of the project as to de novo production of the four dNTPs and Pfu polymerase that are used in NAA techniques, our brainstorming came down to the different versions of how AdAPTED could be implemented in the real world. It is important to note that the production of the two reagents comes from two different plasmids and thus different cultures, and also that the two products are the result of different protocols and demand different handling. Therefore, the implementation of each reagent will be discussed separately, although there are some scenarios that can be used for both of them.
dNTPs
Considering affordability and sustainability as the key factors for the implementation of the project it was decided to provide either lyophilized recombinant plasmid that encodes the enzymes for the production of dNTPs or the lyophilized lysate of the transformed cells that produce dNTPs. Lyophilization is a method for freeze drying that removes the water from the system and allows for room temperature storage. Alternatively, the dNTPs can be produced in a centralized facility and provided to the end user, in a conventional implementation approach.
1. Lyophilized recombinant plasmid
In the first option, the user would have to resuspend the plasmid and use it to perform the transformation. After that, the transformed cells would be grown and the production of dNTPs would be induced using IPTG and AraC. For this scenario, we would have to create and provide the user with an empirical reference curve for the dNTPs’ concentrations versus the inducers’ concentrations versus the culture growth time, to identify the culture conditions in order to yield the requested concentration. After that, the user would centrifuge the cell culture for 5 minutes at 1000 x g (approximately 2000 rpm) at 4°C to cause cell lysis and enable the release of the dNTPs. The produced lysate would be finally used to perform the NAAT. This process is encouraged to be performed in batches and to keep a stock of the produced dNTPs at -20°C, as it is a laborious protocol. The plasmid and the cells can be also stored as stock to repeat the process again. For this scenario, it would also be helpful to provide the user with a positive control, which would be a small quantity of pre-made dNTPs, so that end users will be aware of the expected results after the transformation.
2. Lyophilized cell lycate
In the second option, the user would have to resuspend the lysate and perform the NAAT using a suitable concentration of the lysate, that would correspond to the desirable concentration of the dNTPs. Again, the empirical reference curve should be used during the growth of the cell culture, before providing the lysate to the user, in order to standardize the dNTPs’ concentration in the lysate.
3. Isolated and purified dNTPs
As for the third option, the user follows the established process as with other commercially bought reagents. The production would take place in a centralized facility where the transformed cells would be grown in industrial bioreactors and the production of dNTPs would be induced, using the reference curve. The cell lysis would be performed as described in the first scenario while the isolation and purification of the reagent would be achieved by performing HPLC, as it is described in the protocol that we have prepared for measuring the concentrations of dNTPs. This protocol is described in detail in the Protocols page.
Pfu polymerase
In order to provide the end user with the Pfu polymerase produced as it is described in this project, there are two different scenarios. Similarly to what has been described above, the first option is to provide the lyophilized recombinant plasmid that encodes the polymerase and the second one would be to provide the isolated and purified reagent.
1. Lyophilized recombinant plasmid
The first scenario would be implemented in a similar way as the corresponding scenario for the dNTPs. The user would have to consecutively perform plasmid resuspension, transformation, cell culture using the provided empirical reference curve for the Pfu concentration versus growth time, in order to yield the requested concentration. After that, the isolation of the polymerase can be achieved by heating the bacterial lysate containing the polymerase at 94°C for 5 minutes, causing the denaturation of every bacterial protein other than the thermophilic polymerase of interest. The resulting thermo stable Pfu DNA polymerase is then separated from the other debris of the denatured proteins by simple centrifugation, (Siva Sankar et al., 2019) and used in the NAAT of interest.
2. Isolated and purified Pfu polymerase
In the second scenario, again, the user follows the same established process as with other commercially bought reagents. The production would take place in a centralized facility where the transformed cells would be grown in industrial bioreactors and the cell lysis and isolation would be performed as described in the previous scenario. An alternative method for the isolation of the protein would be using gravity flow column chromatography with a polyhistidine tag attached to the protein, likewise the experiments we performed to identify the produced protein. The process is described in depth in the Protocols page. However, it has to be mentioned that His-tag purification protocol is more expensive and economically unsustainable.
In contrast with dNTPs purification, it is not possible to provide Pfu polymerase to the end user in the form of lyophilized cell lysate, as there are proteases and the enzyme would be denatured fast.
Final Thoughts
Ultimately, in each case there has to be an explicit description of the system that each end user will be provided with, as each one of the possible implementations calls for a different level of effort from the user and different storage conditions, while resulting in different reagent purity.
The lyophilized plasmids scenario for dNTPs and Pfu polymerase is a laborious and time consuming process on the user’s end, but its great advantages make it an attractive alternative for some potential users. In this scenario, the lyophilized plasmids are transported at room temperature, without the need for low temperatures and the user only needs to acquire the system once, significantly reducing the cost compared to traditionally buying the reagents separately. After that, they can either freeze the plasmid after resuspension or store the transformed cells and produce the reagents regularly, depending on their needs. This scenario is expected to be of interest to doctors and nurses who work in remote and isolated settings, where there is a high need of qualitative diagnostic tests and the supply of such reagents is restricted. For example, if an epidemic breaks out in a small and isolated city of a developing country, the financial restrictions and the restrictions related to the transportation conditions of these crucial reagents’ supply might hinder the tracking and thus the cure of the disease.
On the other hand, the lyophilized cell lysate scenario for the dNTPs is less demanding for the user, also sharing the room temperature transportation conditions advantage with the previous scenario. However, the lysate has to be acquired for each use and can not be reproduced at the point of need, the same way as all the conventional reagents. Still, this system is expected to cost significantly less than acquiring isolated and purified dNTPs. The product of this scenario could be used for educational purposes, in Universities and/or schools where students learn about NAATs and are taught how to perform them, without stressing over the purity of the NAAT product.
Finally, the scenario with the isolated and purified dNTPs and Pfu polymerase is a simplified and straightforward process for the end user that values the purity of the reagents. However, this scenario requires that the transportation and storage of the reagents is carried out at -20°C and of course it can not be reproduced at the point of need. For these reasons and also because the isolation and purification process utilizes expensive technology and equipment, this scenario is anticipated to be the least affordable for the end user. The end users of this scenario would be researchers that use NAAT as part of their experimental procedures and want to further use the NAAT product, without compromising its purity and yield.
All the alternatives scenarios for the implementation of the project are summarized in Table 1 with the respective advantages and disadvantages compared to each other, as well as their potential users. It is worth mentioning that the products that correspond to each potential user are not limited to them, as this analysis serves the purpose of comprehension and simplicity. Our team would be more than happy to be able create different products that meet different users’ needs.
| Implementation scenario | Advantages | Disadvantages | Potential uses |
|---|---|---|---|
| Lyophilized plasmids (dNTPs, Pfu) |
Room temperature transportation Can be reproduced at point of use Most affordable |
Requires laborious process | Diagnostics in remote settings |
| Lyophilized cell lysate (dNTPs) | Room temperature transportation | Can not be reproduced at the point of use | Educational purposes |
| Isolated reagents (dNTPs, Pfu) | Simplified use Pure reagents |
Can not be reproduced at the point of use Storage at -20oC Least affordable |
Research labs |