FOR THE FULL EXPERIENCE PLEASE USE A PC :)

iGEM_Athens_2021_AdAPTED

Human Practices

Synopsis

The PCR technique has been, over the years, a necessary tool for research, diagnostics and especially this past year due to the rise of the COVID-19 pandemic. A simultaneous, worldwide rise in demand for PCR procedures has indicated a clear problem regarding the accessibility and production of its raw materials. These problems can be attributed to dNTPs and DNA polymerase. Because their production depends on a centralized industrial market, their total cost is high while at the same time they are synthesized by complicated, low-yield, unsustainable chemical processes [1]. Moreover, PCR techniques are feasible only in centralized laboratories by small groups of lab experts and expensive equipment.

When brainstorming on our project, our initial aspiration was to overcome the above restrictions and enable the decentralization, accessibility and standardization of PCR and other NAA techniques around the world. However, 2020 has taught us from early on that insufficient testing has a direct impact on people: not only on those being tested but also on the ones responsible for the testing process. Therefore, it was impossible to settle on a solution without interacting with the individuals and communities most affected by the problem, since it is only them who can confirm or redirect our theoretical approach. In this context, human practices were a crucial part of our project all along, through which we aimed to fully grasp the above-mentioned problem and design a realistic solution that will offer genuine help to humanity. Our decision-making process was based on receiving information from the right people and communities. Viewing our project through their lens helped us delve deeper into the values we want to invest in and represent through AdAPTED.

1. Awareness and Sensitivity

a. Literature and data

Our initial approach was to review the existing literature on the issue we aimed to address. Through our research, we came across three papers that played an important role in shaping our project [1-5]. Thοse papers introduced us to the importance of considering the accessibility, sustainability and simplicity of our proposed system and were also crucial for our project design.
To confirm and reinforce those findings with additional data, we searched for an analytical report of the commercial production of our reagents of interest. dNTPs are typically manufactured from deoxynucleosides or deoxynucleoside monophosphates (dNMPs) by either chemical phosphorylation or enzymatic synthesis. Our research brought up two major limitations of the chemical manufacturing process: its environmental footprint, and high cost due to its low yields in both the synthetic reaction and purification processes. As for the enzymatic method, it is indeed more efficient yet totally dependent on industrial facilities [6-8]. After validating our initial hypothesis, we then knew we had to ensure the affordability, eco-friendliness plus accessibility of our alternative proposal

b. Shared experiences - Interviews

After having strengthened our theoretical background, we were inspired to be involved in the troubleshooting more personally. We then decided to engage with the people that motivated us the most to learn more about their experiences. First, we contacted the authors of the above-mentioned publications to further discuss the challenges they faced during their research, but gradually more influences were added to the finalisation of our ideas.

Dr. Apostolos Alissandratos:

When researching for our project during the preliminary stages of our brainstorming, we came across Dr. Alissandratos' paper, titled “DNA amplification with in situ nucleoside to dNTP synthesis, using a single recombinant cell lysate of E. coli”. Dr. Alissandratos played an important role in our decision-making, and through our interaction we gained invaluable feedback for many aspects of our project design. Below we document his contribution to both our general inquiries and wet lab-related dilemmas.

Dr. Sanchita Bhadra:

We were intrigued by the way the lack of accessibility and other challenges of low-resource settings are being approached in the paper entitled "Cellular reagents for diagnostics and synthetic biology" written by Bhadra et al. Thus, we contacted Dr. Bhadra in order to learn more about her experience on lyophilizing the end product, as we aspired to do the same. Our conversation helped us understand how to address the issue of accessibility through the way our end product will be distributed to the labs.

Dr. Joseph Fitzgibbon:

The paper written by Dr. Fitzbiggon and his colleagues is entitled “Laboratory Challenges Conducting International Clinical Research in Resource-Limited Settings” and focuses on HIV-related clinical trials. Thus, we contacted Dr. Fitzgibbon, as we were immediately interested in knowing how research challenges are being approached in those settings. Dr. Fitzgibbon shared his experience on this with us, which was very helpful in our deeper understanding of the problem we aim to address: the enhancement of diagnostics in remote settings through the decentralization of laboratory consumables, specifically the PCR reagents.

Talk with "The Internationals":

A pretty unexpected but ultimately insightful conversation with “the Internationals” helped us gain a full picture of diagnostics in remote settings. “The Internationals” are five biomedical students of Vrije University, Amsterdam who volunteered in the Hospital of Moshi, Tanzania in summer 2021. Through an in-depth narration of their eye-opening experience, Iokasti and Chrisa provided us with essential information about the restrictions of medical equipment and consumables in this region.

c. Survey

In order to gain information about the needs of the scientific community and direct our project to those needs, we decided to create and distribute a survey. Based to the answers, we collected data to ensure our project provides a solution to important issues that are present in laboratories all around the globe. The survey was distributed in order to include scientific laboratories in remote areas, biotechnological companies, remote diagnostic centers and also university laboratories and iGEM Teams all around the globe.

2. Guided Design

Our next step was to build upon our obtained knowledge and construct our project design based on the information we gained through the previous steps. Every interaction we had resulted in providing us with valuable technical and general advice at this point and helped us overcome the issues and dilemmas we faced during this process. Also, we followed the directions that were pointed out through the answers of our survey. To see how we AdAPTED, click here.

3. Implementation and adaptation

The last step of our human practices approach was to implement and reevaluate our project idea. People who played an important role at this point are Dr. Koutsioulis and Dr. Topakas and his team.

Dr. Evangelos Topakas:

To test if our designed system can be applied in practice, we decided to perform part of our experimental process in Dr. Topakas’ Lab of Industrial Biotechnology and Biocatalysis at the School of Chemical Engineering of the National Technical University of Athens.
Dr. Dimitris Koutsioulis:

A turning point in the further development of our project was our interaction with Dr. Dimitris Koutsioulis, Chief Executive Officer at EnzyQuest, which is the first certified company for enzyme production in Greece. Dr. Koutsioulis’ research interests and main specialty are in the field of Enzyme Biotechnology. Dr. Dimitris Koutsioulis provided us with invaluable and spot-on feedback not only for the technical aspect of our project but also for its future prospects.

Integrated Human Practices

Each of the previous information was ultimately interconnected and integrated in our project design after thorough consideration and decision-making. Below we document -in bold- how we processed new knowledge, embraced worthy thoughts, compromised, and finally adapted.

Guided Design

Dr. Apostolos Alissandratos

Inspiration and general facts about his research:

Dr. Alissandratos informed us that although prior to the pandemic they had plenty of dNTPs, last year’s events led to a significant decrease in their dNTP stock. In general, in Australia the import of dNTPs was more unrealistic, with cold storage required and instability of the reagents, therefore they resorted to finding new ways of local production of dNTPs. With the production of dNTPs in each lab, the limits to using PCR are widened, especially for developing countries.

In his paper, they present an enzymatic way of producing dNTPs using a deoxynucleoside kinase (dNK). During their research, they found the non-enzymatic way problematic. The chemical production of dNTPs lacks specificity, due to the high complexity of the reacting molecules, such as reactants required for phosphorylation. Another problem present during the chemical production of dNTPs is hyperphosphorylation, which results in toxic products for polymerase, due to inhibition. Lastly, this method requires the use of toxic solvents and extensive purification and isolation. On the contrary, with the enzymatic production of dNTPs, many of the above-mentioned problems could be solved. The specificity increases with the introduction of enzymes, and the products are not toxic for the cell. In general, he underlined the importance of integrating environmentally friendlier techniques in the lab, as well as green chemistry applications in biocatalysis. When choosing biocatalysis, his lab manages to avoid toxic chemicals, lower thermal and pressure conditions, resulting in more sustainable production methods.

We realised that our research ideas were parallel, except for the fact that they didn’t take advantage of the path of de novo dNTPs production in their methodology. However, he confirmed our initial design based on the enzymes of RNR and TSase and reinforced our approach with the following information.

Wet Lab-oriented discussion based on our questions:

a. Selection of polymerase:

Regarding the selection of the polymerase, he informed us that they chose to use Pfu polymerase because of its high fidelity. We were inspired to use Pfu polymerase for the same reason for our design.

Tip: Use of His-tag in the polymerase to confirm its expression with Ni chromatography and facilitate its purification.

b. Plasmid design:

We were also curious to know whether they used separate plasmids for the pfu and dnk genes. He answered that at their lab they used separate plasmids for the DNA polymerase and the dNK enzyme responsible for the dNTPs production, but he suggests that we design only one plasmid that consists of rnr, thyA and pfu genes. Having in mind the effect the size of the plasmid would have in the success of our experiments, we decided to design separate plasmids, one that includes the pfu gene and a second one that includes rnr and thyA genes.

Tip: Insertion of an RBS region before nrdA and nrdB and inclusion of a ~20-30 base gap in between.

c. Strain Selection:

For their experiments, they used E. coli BL21. This was due to the fact that it has a T7 promoter and star D3 specifically was used because of its high mRNA half-life time. Regarding our idea to use E. coli K12, he stated that we need to use a different promoter but this will not have any effect on PCR. He also informed us that MRE600 were used for nucleotide production but it is not sure that they will be helpful for our goal. Based on this conversation we decided to use E. coli BL21 too.

d. Purification of the produced dNTPs:

The complete purity of dNTPs is not a necessity in order to prove our point. It is more important to focus on the accessibility of our method/product. During the lysis of the cells, we have to use a phosphate buffer, in order to avoid the dNTPs hydrolysis by the phosphatases. He suggests the usage of mass spectrometry to check our products. He also stated that ideally, the best is to have a 1:1:1:1 dNTP pool ratio, but the first step is to do the negative control with the E. coli wild type. For diagnostic tests specifically, the ratio is not that important as long as amplification is performed. As a result, this amplification can be non-specific. We keep in mind these statements for future implementation.

Tip: A problem we might face is that dNTPs can be proved to be toxic for the cell. A solution to the toxicity problem is to use lac operator and have the cells grow until they reach the exponential phase. We thus included this tip in our design.

e. Selection of the end-product:

Dr. Alissandratos put emphasis on the fact that PCR systems using the required master mix can be very complex for users and require prior knowledge. This problem is especially prominent in diagnostic centers in decentralized locations, where many of the staff are non-specialized and the number of tests is significant. PCR can be complex even when a pure mixture of dNTPs is used.

Tip: He suggested that by providing a transformed bacterial culture or a plasmid, the application of this technique could be more accessible. Another added benefit is the easier transportation of the DNA, as it does not require cold storage. Lastly, the use of only the plasmid could be easier.

f. Additional technical advice for our project design:

Tip: He advised that we compare our system with the natural dNTP pools in the wild-type E. coli (negative control) in order to confirm the increased dNTP production in our transformed cells.

Goal: To perform a PCR using the dNTPs that were produced through our designed mechanism (maximum 0.1 uL of cell extract).

Dr. Sanchita Bhadra

The initial inspiration of the paper was to find a cheaper source of biological reagents that can be produced locally in research or education areas. Their lab works a lot on polymerases and they constantly transform E. coli cells to overexpress proteins. They use crude lysate of bacteria and because they wanted a way to stabilize the bacteria, they tried lyophilization of the bacterial cells. To test their final product, they collaborated with labs in remote settings. The result was that the lyophilized bacterial lysate survived several months and thus their protocol worked. According to Dr. Bhadra, the most significant advantage of cellular reagents in terms of accessibility is that they can be stored without cold storage of any kind and they require very little instruments and very little training for their production. Working on a small scale for your experiments for your lab using cellular reagents is better in comparison with the usage of purified enzymes because there are a number of steps involved in the purification process.

Regarding the selection of our end product in terms of affordability and accessibility, we had two options for our proposed end-product: either the open outsource of the plasmid or the lyophilized lysate of the cells. Dr. Bhadra advised us that if we use the first option it will be good to have a positive control inside our kit, which will be premade setup reagents so that people will know what to expect when they finally make the cellular reagents. But the final choice depends on the people we send it to, because different users may have different needs. We finally ended up investing in the flexibility of our proposed solution and having a solid plan for each of the two end-products.

Dr. Joseph Fitzgibbon

Dr. Fitzgibbon informed us that due to shipping delays it usually takes longer to get the reagents they need and when they get them, they have limited time to expire. This is also an important financial issue for the lab and this situation got worse because of the pandemic. To address this situation the labs have to validate the systems they work on, perform proficiency testing for all the tests and report their results. He mentioned that growing bacteria to get the reagents also has limitations with the expiration dates, but it is easier to grow them any time rather than wait for an order. This enhanced our decision of maintaining the plasmid distribution and bacterial transformation as an end-product option. He proposed that we should also focus on other amplification techniques such as LAMP or RPA.

Talk with "The Internationals"

Moshi is a small town in Kilimanjaro Region and has a catholic hospital, while the whole region has three to four hospitals in total. The students mostly described to us their volunteering experience in the hospital laboratory, where the technical equipment was more advanced than the average hospital facilities. Hematological, microbiological, and biochemical lab testing was conducted with the highest frequency using blood sampling. In general, only the absolutely necessary lab tests were performed, and in most cases, coronavirus antigen testing was also excluded due to high expenses. Antibody blood testing was preferred for almost every case of common pathogen detection - like HIV and Malaria - while Salmonella was detected through antigen blood testing. However, the ELISA technique was only used in a single hospital in the whole region. As for PCR, it was to be found only in two special centers in the capital of Tanzania and Arusha respectively.

Another significant observation students elaborated on had to do with the scientific background and expertise of the hospital personnel. In many cases, lab technicians lacked the theoretical knowledge and practical skills to operate lab equipment, such as the laser microscope. Another issue was about the interpretation of lab test results, where difficulties were also apparent.

Last but not least, we discussed availability issues with an emphasis on the shipping of materials. The students informed us about a 2-week delay in the shipping of specific buffers in contrast to the constant availability of the most highly used diagnostic tests. However, Iokasti pinpointed that the main problem with diagnostics in Moshi was people’s ignorance of having to be tested regularly. This overweight the absence of materials in the hospital, and thus girls advised us to put great emphasis in science communication of frequent and spot-on diagnostic testing.

After considering the advice of Dr. Fitzgibbon and the Internationals, we had the idea of visualising a widely used diagnostic technique for the detection of COVID-19 in point-of-care facilities in a simplified step-by-step guide. LAMP was chosen due to its suitability and efficiency in such settings and the minimum equipment needed, as Dr. Fitzgibbon indicated [9-13]. This way we hope to facilitate not only the implementation of its experimental procedure but also the interpretation of the results by the personnel in charge. Ideally, our produced dNTPs and DNA polymerase could be included in the LAMP Mastermix, along with salts, four to six specific primers, an additional heat-stable reverse transcriptase for the detection of RNA viruses and a pH-sensitive indicator dye (cresol-red).

Survey

In order to ensure the accessibility of our system, we have the ambition to transfer it to different settings where PCR reagents are used and evaluate our end-product under real-life conditions. These settings would include healthcare facilities and diagnostic laboratories in Athens, Community Health Centers in outermost regions and island areas across Greece, but also academic institutions and High Schools that use reagents for research and educational purposes.

During our brainstorming, receiving critical feedback from multiple targeted users regarding our project implementation helped us tailor our end-product to the actual needs of those who will benefit from it. In order to upscale our project from a theoretical frame to reaching its actual impactful potential, we carefully selected and distributed our survey to the following potential end-users of our project:

Researchers that collaborated with us from laboratories in Greece and abroad.
Laboratories in island areas in Greece.
Laboratories in the outermost regions of mainland Greece.
Private diagnostic centers in Athens.
Laboratories of Biotechnology companies in Greece and abroad.
Academic laboratories that use PCR for educational purposes.
Students from iGEM Teams all over the world.

a. dNTPs:

In total, 24 lab representatives took part in the survey and almost all of them use dNTPs for their experiments, in techniques such as PCR, RT-PCR, RT-LAMP, real time PCR, qPCR, ligation, genomic, diagnostic PCR tests, in vitro transcription, library construction for sequencing and plasmid engineering. Most of them answered that these techniques require skilled personnel, but there are no difficulties regarding the storage and handling of dNTPs. The order frequency is once every three months. On the question of whether there were any problems regarding the availability of the reagents or not during this year, the answer was not clear (66,7% I don’t know, 25% No and 8,3% Yes). Based on their annual budget, dNTPs are of medium cost and their purity is highly important for their experiments.

1 / 7

2 / 7

3 / 7

4 / 7

5 / 7

6 / 7

7 / 7

❮ ❯

b. Selection of the end-product:

Our research shows that the idea of each lab producing their own dNTPs using a streamlined and eco-friendly protocol is appealing to the end-users. The idea of distributing dNTPs that are stored in a lyophilized lysate of E. coli cells that will be stored in root temperature was also attractive. Between the two of them the first one was more attractive as shown in the results below. The research showed that other methods such as diffusion were not appealing, because the production will be slower.

Despite the fact that maintaining flexibility of our-end product was of major significance to us, we wanted to take into consideration the survey’s results. Thus, we wanted to ensure that the design of the experimental procedure of transformation would be as optimized as possible and the end-product purified enough. However, the purification process was not as simple as we initially thought it would be, thus we attempted to describe the methodology as clearly and well-defined as possible.

1 / 4

2 / 4

3 / 4

4 / 4

❮ ❯

c. DNA polymerase:

According to the survey’s results the most commonly used DNA polymerase is Taq and the second one is Pfu polymerase. We chose to use Pfu polymerase for our system because of the conversation we had with Dr. Alissandratos, who mentioned that Pfu has high fidelity and applied better in diagnostics. Also, the fact that Pfu was characterized in the iGEM registry was an extra advantage. In addition, it is obvious that DNA polymerase is an important tool for their lab and the idea of producing it in their lab using a streamlined and eco-friendly protocol appears to be highly attractive to almost all lab representatives that took part in the survey. This resulted in our decision to include DNA polymerase in our design. Also, purity is something that we had to keep in mind as shown by the results.

1 / 4

2 / 4

3 / 4

4 / 4

❮ ❯

Implementation and Adaptation

Lab of Industrial Biotechnology and Biocatalysis, NTUA

In order to test whether our designed system can be applied properly in practice, we performed part of our experiments, specifically the transformation, expression and purification of pfu polymerase, at Dr. Topakas’ Lab. In addition to our successful results, we also had Dr. Topakas’ confirmation that our proposed steps to the end-product are realistic and can be implemented in practice. They also stated that our methodology could be an actual alternative to be used in their lab, which was a hopeful validation of our overall work.

Dr. Dimitris Koutsioulis

a. Pfu:

By the time of our meeting, we had already settled on using pfu polymerase in our experiments, due to its high thermostability and proofreading capacity (low error rate) in comparison with other alternatives. However, Dr. Koutsioulis informed us about a more widely commercially available variant, Phusion DNA polymerase. Phusion polymerase is a novel pfu-like polymerase with a processivity enhancing domain, which presents higher efficiency and accuracy than the single enzyme. After our conversation, we considered his suggestion of turning to a more widely preferred enzyme and decided to modify our genetic circuit in order to include Phusion polymerase instead. Therefore, even though our initial measurements derived from the expression of pfu from the transformed strains, we also decided to include a plasmid construction with Phusion polymerase for future use in commercial applications, as shown below.

b. dNTPs pools:

Dr. Koutsioulis also emphasized that we should maintain the dNTPs ratio to 1:1:1:1, as it is critical for error avoidance during PCR, which complies with our literature findings. However, he suggested that we should focus on regulating the dNTP pools only after we make sure our system’s functionality and efficiency are guaranteed.

c. Purification process:

Additionally, he highlighted the importance of the purification of dNTPs and DNA polymerase as the crucial step which will determine the “attractiveness” of our proposal. He suggested that we develop a very well-defined purification protocol with a strict selection of our used materials in order to reduce any time-consuming, complex, and costly intermediate steps. This tip significantly defined the documentation of our experiment protocols. As a future redirection of the project, he proposed that we examine the possibility of using transmembrane transporters to easily extract the desired enzymes [14].

d. Streamlined process - Cost - Scaling up:

Regarding the future plan of the scaling up of the process, he advised that we should compare the steps of the chemical production with the desired streamlined production of the dntps and DNA polymerase. Constructing a flowchart of our method will also define the total cost of our alternative. He also underlined the importance of the technical equipment required for the intermediate steps as the determinant of the total cost. This statement refuted our first impression that the cost of the process depends mainly on the raw materials used. Also, he mentioned that the purification process required to reach the final products is the most difficult part of the scaling up and for that reason it has to be well-designed and studied. All this information will be helpful on the scale-up part of the process in the future.

Team:Athens/Human Practices

FOR THE FULL EXPERIENCE PLEASE USE A PC :)

Synopsis

1. Awareness and Sensitivity

2. Guided Design

3. Implementation and adaptation

Integrated Human Practices

Guided Design

Dr. Apostolos Alissandratos

Dr. Sanchita Bhadra

Dr. Joseph Fitzgibbon

Talk with "The Internationals"

Survey

Implementation and Adaptation

Lab of Industrial Biotechnology and Biocatalysis, NTUA

Dr. Dimitris Koutsioulis

igemathens2021@gmail.com