1.1 Project Summary

The Ecuadorian amazon region stands out for its great biodiversity, extended watersheds and tropical landscape. Napo is a territory known for its ancestral indigenous communities, biodiversity, and fluvial systems, which are endangered mining activities and lack of knowledge of remediation technologies. Current studies revealed the presence of heavy metals in Napo’s rivers due to mining activities [1], where copper was found in concentrations 500 times higher than the allowed limit. The presence of heavy metals in Amazon water sources has a negative impact on ecosystems and population, due to its consumption without previous treatment in rural and urban areas. Heavy metals are highly toxic, non-biodegradable, and are able to accumulate in plant and animal tissues. Copper (Cu) [2], among other metals, is also associated with the inhibition of growth development in plants and for population when consumed through contaminated water and food. Also, the long exposure of Cu causes health problems in the population. 

Ikiam university is located in the Napo province, and it's focused on the investigation and generation of ideas and projects related to solving many of the issues that affect the Amazon region. The present project focuses on using and exploiting the advantages of synthetic biology in order to apply it to bioremediation approaches on areas affected by anthropogenic activities. The SynBio Ikiam project is focused on presenting a green alternative for the detection and removal of copper from the environment, as a priority for the wellness of the Amazon Region.

Some microorganisms are capable of achieving a metabolic process called bioleaching, where heavy metals can be used as a source of energy in order to satisfy their metabolic needs. This is done by the absorption and transformation of the element into a more soluble form [2], that can be easily removable through refinery approaches allowing its purification. Bioleaching stands out for its efficiency in the removal and separation of metals such as silver, uranium, iron and copper, allowing its sustainable recovery with no environmental implications. For these reasons, the present project proposes the design of plasmids for the adaptation of Escherichia coli to water contaminated with Cu and gives it the capacity of bioleaching for this heavy metal. In this way the Amazon environment could be restored and Cu could be reused.

Our design involves the detection and bioleaching of copper-contaminated water samples through the modification of the E. coli bacteria. The module for the detection of copper is based on the CusS-CuSR two-component system that allows the transport of extra cytosolic copper. The promoter CusC allows the transcription of RBS followed by a GFP that emits bioluminescence in the green zone on the visible spectrum. 

With respect to the adaptation of E. coli to the acidic and toxic environments produced by Cu, biological parts were taken into account that help the neutralization of reactive oxygen species (ROS) that are generated by the stress of metals in the environment. For this we plan to implement parts such as the sodA (converts O2- to H2O2) and katG (converts H2O2 to H2O + O2) genes that transform free oxygen ions to less harmful species such as water. Another mechanism to be used for detoxification is glutathione encoded by gshA which with the help of glutathione peroxidase (btuE) and glutathione reductase (gor) expression will allow the removal of peroxide radicals [3]. In addition, for copper uptake the design is intended to knock out genes belonging to the CusCBA operon that encode efflux pumps to prevent the export of Cu from the periplasmic medium of the bacteria to the outside, while overexpressing Cu ion importers (copC and oprC) and inner membrane transporters (copD and hmtA). 

Together, each of the parts of our biological circuit will allow E. coli to detect the copper present in water samples and subsequently bioleach it. Importantly, the matching module parts confer tolerance to harmful conditions such as high acidity and heavy metal concentration. Therefore, the removal and accumulation of copper contaminants will be possible and achievable through environmental-friendly approaches.

References

[1] Capparelli MV, Moulatlet GM, Abessa DM de S, Lucas-Solis O, Rosero B, Galarza E. An integrative approach to identify the impacts of multiple metal contamination sources on the Eastern Andean foothills of the Ecuadorian Amazonia. Science of The Total Environment. 2020;709: 136088. doi:10.1016/j.scitotenv.2019.136088

[2] Giachino A, Focarelli F, Marles-Wright J, Waldron KJ. Synthetic biology approaches to copper remediation: bioleaching, accumulation and recycling. FEMS Microbiology Ecology. 2021; 97. doi:10.1093/femsec/fiaa249

[3] Rademacher C, Masepohl B. Copper-responsive gene regulation in bacteria. Microbiology. 2012;158: 2451–2464. doi:10.1099/mic.0.058487-0

1.2 Promotional Video 

1.3 Project Presentation Video

1.4 Education and Communication Infographic.

1.5 Team and attributions

2.1 Design Roadmap

2.2 Excellence in Biological Engineering Design

2.2.1 Standardization.

-Biosensor

Our first module was a biosensor; it was composed of two biological parts. The first part has an activator CueR stimulates a transcription copAp promoter and constitutive promoter-RBS, the second parte has a promoter inducible called CopAp is a Cu(I)-translocating P-type ATPase ( Gregor y Rensing, 2001), 5 UTR, sfGFP and a terminator.

The parts were chosen by the team of Team Bielefeld-Cebitec (BBa_K1758320, BBa_K1758323), because these pieces show significant results in vivo. The CopA (R. metallidurans) is regulated by CueR.

Fig 1. Plasmid assembled for Golden Gate

- Coupling of copper importers

The iDLBB_001952 composite part is made up of two importers on the outer membrane, CopC and OprC, as well as two transporters on the inner membrane, CopD and HmtA. Together, the role of these proteins is to promote copper uptake from external importers with the help of domestic importers.

The native origin of each part is Pseudomonas brassicacearum 3RE2-7. Both CopC and HmtA are regulated by the T7 promoter and the T7 TE terminator. On the other hand, CopD and OprC are regulated by the pBAD / araC promoter and a double terminator. Vector pSB1C3 was used for transformation.

- Knockout of the CusF gene

Cu export occurs from the cytoplasm back to the periplasm via the P-type ATPase, CopA, and is transferred to a periplasmic chaperone, CusF, which can then deliver it to the periplasmic cuproproteins or CusCBA efflux pumps for excretion . Therefore, to avoid the exit of Cu, the silencing of the CusF protein will be carried out, using the Lambda Red recombination system. The part BBa_K3076500 already available in iGEM was used.

- Toxicity adaptation

The module of adaptation was based on Team Bielefeld’s design; this team participated in iGEM 2018. It has the ability to degrade superoxide anion and its by-products such as hydrogen peroxide into non-toxic forms preventing cell death in the presence of copper. In addition, tolerance was found to increase up to concentrations of 8mM CuSO4 for a period of up to 9 hours. This design had an effective fluorescent response as in vivo such as in vitro. Also, their data show the correlation between the emitted fluorescence and the different concentrations of copper.

Those parts used were BBa_K2638118 that has a superoxide dismutase gen (sodA) and a catalase HPI gen (katG); these genes are used to dismutate hydrogen peroxide in water and oxygen. It can be used to fight ROS. And the other part used is BBa_K2638112 that has glutathione reductase gene (gor), glutathione synthetase gene (gshB) and glutathione peroxidase gene (btuE); this system uses glutathione as a substrate to scavenge reactive oxygen species. As a byproduct, disulfide glutathione is being generated. So we assembled these two parts BBa_K2638118 and BBa_K2638112 in one piece.

2.2.2 Optimization.

-Sensor of copper

After being reviewed references the different parts biological by iGEM groups we decided for two parts because they have efficientes promoters, RBS and terminators for detecting of copper in our system.

- Coupling of copper importers

Our optimization was performed based on the results obtained by previous iGEM groups. In the case of importers, the promoters that showed the best results were chosen, which are T7 for CopC and HmtA, and pBAD / araC for CopD and OprC. Additionally, spacers, the T7 TE terminator and a double terminator were added.

- Knockout of the CusF gene

As already described above, it was chosen to block the CusF gene. Another alternative to ensure that the copper does not leave the periplasm was to block the entire CusCBA efflux pump; however, the results shown by an iGEM team in 2019 showed that there were no significant differences when doing it. On the other hand, after blocking the CusF gene there was a greater accumulation of copper.

- Toxicity adaptation

Our optimization in this module is to assemble two ROS mechanism performers before in one piece under the control of one promoter. Therefore, the module consists of a superoxide dismutase A (sodA) and catalase (katG) under the control of a strong RBS and also consists of a glutathione synthase (gshA), glutathione peroxidase (btuE) and glutathione reductase (gor) under the control of a strong RBS, these two parts are linked by a spacer and under the control of a pBAD promoter in a plasmid pSB1C3.

2.2.3 Build and Test

The development of the Synbio Ikiam project was based on the generation of 3 biological components: biodetection, bioleaching and survival. Once the design of each component was completed, we proceeded to the synthesis of each fragment and the construct. In order to evaluate the state of transformation of each component, we chose to use the vectors pKD46 (Bioleaching) and pSB1C3 (biosensor, Cu survival) for their independent evaluation. Parallel construction of the plasmid assembly will be performed with Golden Gate cloning in order to assess possible mutation scenarios.

The transformation of the parts will be performed according to a protocol already established by the Bielefeld-CeBiTec team, based on the use of thermal shocks in chemo-competent bacteria. In order to perform expression analysis, E. coli strains transformed with one or more combinations of each component will be generated.

Evaluation assay

The evaluation and verification of the transformation process will be performed by two processes:

Antibiotic resistance: Each component of the biological circuit has a different resistance gene

●                  Biosensor: chloramphenicol

●                  Survival: Kanamycin

●                  Bioleaching: Ampicillin

This evaluation was performed in solid LB media supplemented with 1 of the antibiotics and in combination in order to separate the competent bacteria for the process to follow.

Colony PCR: A second confirmation was based on the use of Colony PCR, where primers were designed using the ends of the sequences of each biobrick and the amplification of these was estimated in competent bacteria that had passed the antibiotic resistance evaluation.

After the confirmation, each competent bacteria strain will be stored in a stock sample consisting of a solution of LB medium supplemented with 20% v/v glycerol and preserved at -80

°C.

Efficiency assays

Once we have assessed the desired transformation of our bacteria, a series of evaluation assays will be carried out. Prior to the assays, all transformed bacteria will be previously cultivated in LB media using a rotatory incubator.

In order to evaluate the efficiency of the biosensor component, bacteria will be suspended in a solution with PBS with an optical density (OD) at 600 nm around 0.8-1.3. Then, the bacteria will be submerged in another solution with CuSO4 at different concentrations (1 - 8 mM) with a control sample with no CuSO4. The generated bioluminescence signal will be measured through UV/Vis at 480 nm in order to generate a calibration curve. The generated data will serve as a base for statistical optimization in order to improve the signal generation for its use on field samples from contaminated areas. The survival and bioleaching components will be evaluated using LB media supplemented with CuSO4 at the same concentrations (1 - 8mM) with a control sample with only LB media. Then, the OD at 600 nm will be measured in order to measure the survival rate of the bacteria compared to the control sample.

2.3 Human Practices

Our team has the advantage of belonging to an Amazonian city where there is a repeated coexistence between the city and the indigenous communities of the area, in the same way, being an Amazonian city, there is easy access to various ecosystems found in the surroundings. In Ecuador, mining is an issue that is discussed every day, this in some cities is common to the point that mining is accepted in certain areas. However, in Napo this issue is new for everyone, both for foreigners and for local communities, for that reason when mining began to be heard in Napo, several groups showed their discontent. We as members of the Ikiam University, located in this province, decided to seek solutions for this problem, but without first consulting with the community that makes up the city of Tena in Napo.

Fortunately for our team, during the last few months there have been several meetings of groups that oppose this idea of ​​mining in the region, so it was decided to conduct interviews with several of the participants in these meetings, this with the idea to know their opinion about this practice and also to know what their persecution is about synthetic biology and if there is an acceptance of the project that has been raised for iGem Design League 2021 by the SynBio Ikiam team, which seeks to bioleach copper from polluted rivers in the region by using modified E.coli to meet our stated objective.

With these interviews we realized that most of the participants did not deeply understand what synthetic biology is, but we invited them to follow us on our social networks and with a little explanation they understood and accepted the objective of our project in a good way. On the other hand, some did not feel comfortable with the idea of ​​using microorganisms in ecosystems, they felt more satisfied with traditional methods.

2.4 Integrated Human Practices

Based on interviews with indigenous leaders, we believe that the implementation of Synbio Ikiam directly in rivers contaminated by mining would not be the most appropriate. This is due to the release of GMOs to affected environments, therefore we proposed the generation of a bioreactor which can be implemented by the communities themselves to facilitate access to safe water to their homes. It is also necessary to make strategic alliances with the affected communities to train them in the management of these processes. Finally, it is proposed to use the obtained waste with high copper content as an economic income in the future.

2.5. Impact on the Sustainable Development Goals (SDGs)

Our project can contribute significantly to the specific objectives that we have chosen from the SDGs, since it would directly help objectives 6 and 14, since these are directly related to aquatic ecosystems, the SynBio Ikiam project seeks to clean the Amazonian rivers of Ecuador of copper generated by mining, in the same way, if the project is a success, it could be used as a basis to design more microorganisms that detect and can bioleach more metals, until reaching the point of being able to clean the water of any place Thus our project contributes to the global objectives for sustainable development of the United Nations.

To achieve these objectives, a collaborative work is needed between several teams that have similar objectives, that is why a meeting is held for our collaborations so that both teams know what they have in common. In the first place, it began by looking for teams in the country that focus on the same objectives, but we do not focus that the teams have the same objectives to which we aspire, but rather that they are related, because these would broaden our point of view. about the path we are taking with our project.

We have evaluated the ideas of our project around 3 objectives, 6, 9 and 14, two of them are of utmost importance to us, because one of our ideas is to help the indigenous Amazonian communities of Napo, these communities do not have access to Drinking water, so rivers are their only option and their health would be compromised if they consume water contaminated with copper from mining activities in the area. In addition, the fauna of the rivers is also affected by this excess of copper, which in the long run would represent a problem for the ecosystems of the region.

The impact of our project would be measured quantitatively and qualitatively, for the first results the copper levels would be measured in water samples obtained from contaminated rivers, these samples would be treated with our modified E.coli and later the level would be measured again. of Cu, this would be a great advance for our project, because the functioning of our microorganisms would be evidenced. The qualitative form would be given by the communities and their level of satisfaction when noticing a change in the rivers that surround them.

3.1 Improvement of an existing iGEM design.

The module of Detection was based on Team Bielefeld’s design due to this design having an effective fluorescent response as in vivo such as in vitro. Also, their data show the correlation between the emitted fluorescence and the different concentrations of copper.

The adaptive module is based on parts of the Bielefeld- CeBiTec team that participated in iGEM 2018 as it has the ability to degrade superoxide anion and its by-products such as hydrogen peroxide into non-toxic forms preventing cell death in the presence of copper. In addition,

tolerance was found to increase up to concentrations of 8mM CuSO4 for a period of up to 9 hours.

The bioleaching module is based on parts designed by the Bielefeld-CeBiTec team and the Hong Kong JSS team. We selected the first team because they complemented the native copper accumulation system of E. coli through the expression of outer membrane importers (CopC and OprC), as well as inner membrane transporters (CopD and HmtA), as a result they obtained a system highly efficient accumulation and specific for copper ions. On the other hand, the second team designed double-stranded DNA substrates for Lambda Red recombineering to knockout four major genes involved in the copper ion export pathway in E. coli. We chose this team because they used a feasible system to avoid the export of copper, improving the efficiency of its accumulation.

We planned to improve it by implementing our module of adaptation. To improve the adaptation module, another detoxification mechanism was implemented using a spacer linked to a glutathione synthase encoded by gshA that with the help of glutathione peroxidase (btuE) and glutathione reductase (gor) expression.

Our proposal is to improve the importation of copper ions. To do this, we combine OprC, CopC, CopD and HmtA in one BioBrick. Additionally, we chose the promoters that showed the best results after the team's experimentation and added spacers and terminators. The adaptive module will enable the bacteria to resist toxicity and combat oxidative stress caused by reactive oxygen species ROS. This is through the sodA (O2- to H2O2) and katG (H2O2 to H2O +O2) genes that transform free oxygen ions to less harmful species such as water. Apart from the protein glutathione synthase encoded by gshA which with the help of the expression of glutathione peroxidase (btuE) and glutathione reductase (gor) will allow the elimination of peroxide radicals.

Through our proposal to combine copper importers and transporters into one BioBrick we hope to significantly improve absorption effectiveness. In this way, the bioleaching of copper from copper-contaminated water samples will have a remarkable result.

To test this improvement in the laboratory, E. coli would first have to be transformed with a vector containing the copper importers and transporters. Subsequently, place the transformed bacteria at different copper concentrations and perform absorbance measurements. Due to a greater accumulation of intracellular copper is expected, the result would be reflected in a lower growth of the bacteria due to the toxic effects of copper.

3.2 Collaboration

SynBio Ikiam has few collaborations, but if we have participated in several of them, without a doubt they were all of great importance in shaping our project based on the perspective of other teams that have more experience with synthetic biology. Collaboration with iGem Biotech-EC was of great importance for our team, our collaboration was carried out virtually, in the beginning activities were carried out to get to know each other among the members of each team and what were the objectives of each of our projects, for Luckily, the LATAM FEST activity strengthened our ties and for our last collaboration, more focused topics were discussed and there was an exchange of information between both teams where we supported ourselves in the design of the circuit of each of the projects, each team generated recommendations so that the another improve its design, as a result of these collaborations it was possible to design a better circuit and improve it.

On the other hand, our participation in activities organized by other teams was constant, initially for these collaborations games were used to strengthen relationships between members of various participating teams, this helped us build the basis of the ideas that would form our project This is due to the recommendations made to us by others towards the approach that our project should take.

3.3 Entrepreneurship and Innovation

For the project that SynBio Ikiam has proposed, an entrepreneurial idea has not been carried out in the short term, this mainly due to the security restrictions that limit us, since our project focuses on bioleaching copper from rivers and carrying out this action would have several legal steps that we should continue. However, if we consider an ideal scenario, the undertaking of our team would function with an online service and under recommendation, in the same way our main clients would be mining companies or governments with river bioremediation projects, these two groups would be our most important clients. We would enter the market with strong competition because normally in these years to solve heavy metal problems in rivers physical and chemical methodologies are used, so a SWOT would be necessary to know our opportunities to stand out in the market of companies that are dedicated to solve environmental problems.

3.4 Education and science communication

It should be taken into account that for reasons related to the pandemic, the team could not give face-to-face talks to community members so that they are aware of the ideas proposed by our project, but social networks became our best allies, the Publications that are made on our Instagram and Facebook pages are focused on educating our target audience about topics related to synthetic biology and our project, in the same way the material used are reel images that simplify and facilitate the understanding of the subject by part of the public. Our audience is varied, from young inhabitants of indigenous communities, through professionals who know the subject matter to housewives, that is why our publications are simple and simplified so that they can be understood by anyone who sees our publications.

The graphic material that we use can also be used by outsiders, these can be used as a basis to give an introduction to issues of environmental remediation or synthetic biology. One objective that the team's social media area has set is to create publications in Ecuadorian indigenous dialects, which in the future can be used by team members or outsiders to explain a topic for obvious reasons. country returns to a face-to-face modality in which we as members of SynBio Ikiam can go to educational units, promote the learning of synthetic biology and show how important this subject is for the human being.

Social media links:

https://www.instagram.com/synbio_ikiam/

3.5 Policy, biosafety and/or biosecurity

Since 2008, Ecuador has proclaimed itself as a GMO-free country, prohibiting the cultivation and production of GMOs in national territory. However, there have been certain openings in the entry of GMOs for strictly research purposes. Although this limits the scaling up of Synbio Ikiam's proposal, this project is being promoted as one of many proposals necessary to influence the country's laws to allow its use within the national territory. Although the use of E. coli bacteria does not imply a danger to public health, their transformation and use for biological processes implies the use of biosafety measures.

The main environmental measure of the Synbio Ikiam propostal is the implementation of a bioreactor, which prevents the release of GMOs generated by the project into the environment, thus preventing any environmental implications. Another factor to consider are the necessary measures for the management of waste generated during the process, being in this case rich in copper, which can be sent for refining and obtaining copper. For the management of copper-rich waste generated, strategic alliances are proposed with governmental entities or mining concessions that can be used as raw material for the extraction of this metal.

3.6 Diversity and inclusion

From the beginning the team was focused on being diverse, this was facilitated because most of the team members belong to the Ikiam Amazon Regional University, which is made up of students who come from various parts of the country, for that reason the team was destined To have members belonging to the province of Napo, the region in which our project is centered, this diversity of members allowed us to have different approaches to the ideas of our project and these approaches worked, because they were based on the realities that each one has lived . However, among the members of the team the idea has arisen to include young people belonging to the indigenous communities that are in the area, this with the idea of ​​being able to include these groups to synthetic biology and that their limitations do not stop these young people from Advancing in their preparation as professionals and also their vision of synthetic biology is important for the design of future projects because the approach they would bring would be influenced by their beliefs and customs, which would allow us to efficiently help people from these groups, since the project that will help them was carried out by members of their community. That is the advantage of Ecuador, it may be a small country, but it is diverse in cultures and that diversity has benefited in the design and presentation of our project for iGem Design League 2021.

3.7 Arts and creativity activities and results

Synbio Ikiam participated in an event related to the anniversary of Ikiam University. In order to promote science divulgation and the importance of synthetic biology, we participated in designed activities related to DNA extraction with homemade materials. These methods were proposed for children to perform scientific activities at a low age and also to know more about synthetic biology to a general public. This event was accompanied with ancestral games activities and traditional kichwa dancing performances.

Designed Biological Parts

Safety Form Questions and Answers

PDF/Word Filled Template