iGEM 2023 — Team ashesighana

### Notebook

## GROUNDWORK

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Groundwork

#### APRIL

* The selection process for the 2023 Ashesi Ghana iGEM team started in April 2023, and the final team was chosen on April 11th of that year.

* Final list of the 2023 Ashesi Ghana iGEM team and roles they applied for:
* Anne Achieng - Entrepreneurship
* Anthonio Vera Honam - Entrepreneurship
* Chalton Mandaza - Hardware
* Daniel Byiringiro - Software/Modelling
* Martha Chaitezvi - Dry-lab
* Marvellous Chapfura - Dry-lab
* Maxwell Nyimbili - Web-development
* Mohammed Elmir - Web-development
* Nana Adwoa Ewudziwa Newman - Hardware
* Netanya Anila Bhakta - Hardware
* Tendai Machaya-Software/Modelling

#### MAY

* In May 2023, the newly formed Ahesi Ghana iGEM team met with their PI, Dr. Rosca, and their advisor, Prince Edem Samoh. They discussed the scope of their project options and the expectations for the upcoming iGEM competitions.
  In addition, a specific date was scheduled for the team to begin their preparations for the upcoming iGEM competition.

## JUNE

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June

#### WEEK 1 - JUNE 5th TO 9th

#### MAIN FOCUS OF JUNE: RESEARCH

* We brainstormed project ideas and settled on having the
  project tackle lithium detection, mining, and recycling. Furthermore,
  we began research on how we could use bacteria to facilitate our project idea.
  With regards to safety in handling the bacteria, we concluded that the bacteria should be enclosed
  in microgels. This would allow the bacteria to collect the lithium, for detection, mining, or recycling,
  without leaking into the environment.

* We brainstormed team names and mottos and settled on the name BioLith,
  and the motto “mining lithium the sustainable way”.
  Additionally, we began working on the application for the impact
  grant and the team’s infographic.

![](https://static.igem.wiki/teams/4746/wiki/kick-off.png)

###### Kick-off day in Ashesi University's Bioengineering lab

* BioLith had a synthetic biology tutorial session,
  with the team's PI, Dr Rosca. Thereafter, the team looked
  into lithium-sensing riboswitches to assess feasibility of
  lithium detection, mining, and recycling using
  bacteria. Therefore, the team went through a scientific
  report titled Lithium-sensing riboswitch classes regulating the
  expression of bacterial cation transporter genes by Neil White,
  Harini Sadeeshkumar, Anna Sun, Naarasimhan Sudarsan & Ronald R. Breaker.

* The team was introduced to Benchling, and as an activity, performed
  annotation on the nhaA-I, one of the riboswitches used in the scientific
  report we recently went through. The team was also introduced to restriction
  enzymes: Type I, Type II, and Type III.

![](https://static.igem.wiki/teams/4746/wiki/annotation.png)

###### Gene annotation tutorial session

#### WEEK 2 - JUNE 12th TO 16th

* The team started looking into current lithium mining
  techniques. In addition, the team researched on lithium-pathfinder elements, which
  include Potassium, Calcium, Rubidium, Strontium, Tin, Caesium,
  Rubidium, Arsenic and Gallium. And at this point, the web development
  team, a sub-team of BioLith, started working on a prototype for the
  team's website.

* Development was made with our detection strategy of lithium. We would use
  bacteria that change color in the presence of lithium. We decided that
  the bacteria would change to a blue color. The hardware team, a sub-team
  of BioLith, started working with Raspberry Pi to develop a camera that could
  capture the color change of the bacteria.

* The Dry-lab team, another sub-team of BioLith, looked into genetic engineering,
  Bacteria streaking, Transformation, Agar preparation, Media preparation and Competent cell.
  These are some of the techniques needed to develop bacteria with specified traits.
  The web development team had a prototype for the home page and began working on the Project's description
  page of the website. The team submitted the impact grant application and completed the team’s infographic.
  Furthermore, the team began working on the design for the team logo.

![](https://static.igem.wiki/teams/4746/wiki/logo-brainstorm.png)

###### BioLith logo design

#### WEEK 3 - JUNE 19th TO 23th

* The modelling team started working on how we could
  interpret the pictures captured by the camera, put together
  by the hardware team, to determine the concentration and likelihood
  of lithium presence. The hardware team started working on how to add
  GPS location to the images captured, using the GPS module with Raspberry Pi.
  The web-development team began uploading parts of the website onto iGEM's GitLab
  repository.

* The dry lab team had a tutorial session on pipetting and agar preparation.

![](https://static.igem.wiki/teams/4746/wiki/group-picture.png)

###### BioLith Lab team

![](https://static.igem.wiki/teams/4746/wiki/pippeting.png)

###### Tutorial session with Prince

* The dry-lab team had another tutorial session on cell competency and
  overnight culture of BL-21 cells and AE-Blue bacteria. The AE-Blue bacteria had plasmids
  that gave them the trait of turning blue. The dry-lab team went on and conducted
  sub-culture and then performed mini-prep on the AE-Blue bacteria, to
  collect their plasmids. Furthermore, the plasmids were added to the BL-21
  cells to also give them the trait of turning blue.

* The hardware team were able to link a GPS location to an image taken. The modelling
  team came up with a model using the Python programming language and blue
  pixels to represent bacteria that turned blue, to calculate the probability
  of lithium presence. The entrepreneurship team,
  another sub-team of BioLith, completed the problem statement and our proposed solution. Their next steps
  were then be to generate a SWOT and STEEPLE analysis.

* The web-development team completed the Project’s description page and the home page of the team's website,
  and started working on drafts of the Team page and the Notebook page of the website.

![](https://static.igem.wiki/teams/4746/wiki/gps-module.png)

###### GPS module

#### WEEK 4 - JUNE 26th TO 30th

* All sub-teams: Hardware team, Entrepreneurship team, Dry-lab team, Modelling team and Web-development
  team, gave updates on work completed to that point. Research on lithium mining, biomining and lithium
  detection continued. Furthermore, the modelling team began research on mathematical bioleaching models.
  We also found DNA gene sequences that could be used for the detection of lithium Pathfinders,
  we then put them in Benchling.

* The Hardware team transitioned from using Raspberry Pi to an esp32 cam. Therefore, the modelling team began the transition of writing their algorithm in the C programming language, instead of the Python programming language, to fit the esp32 cam.

* The Entrepreneurship team started looking into the Total Available Market (TOM), Serviceable Available Market (SAM),
  and Serviceable Obtainable Market (SOM) of lithium mining and recycling.

## JULY

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July

#### WEEK 1 - JULY 3RD TO 7TH

#### MAIN FOCUS OF JULY: DESIGN

* During a virtual meeting with our project leader, Dr Rosca, we decided on
  a date for an upcoming meeting with Cultivarium. Cultivarium is a company
  that aims to provide open-source tools for life scientists to access new
  microorganisms, as mentioned in their description. In addition, Dr Rosca sent an email to Atlantic Lithium to suggest potential collaborations.
  Our Entrepreneurship team compiled their research
  into a handbook, which will provide a more comprehensive
  understanding of our business model.

* BioLith Ashesi Ghana was awarded the
  Impact Grant!
  Furthermore, we began generating ideas for the promotional and presentation videos for the BioLith project.

![](https://static.igem.wiki/teams/4746/wiki/notebook/picture4.jpg)

###### The list of the impact grant winners.

* CultivariumBioLith met with Cultivarium, who agreed to help the team with Bioleaching data
  of the Acidothiobacillus ferrooxidans bacteria. In exchange, Cultivarium requested
  two feedback sessions, one in September and one in November, each lasting 30 minutes. Cultivarium recommended showcasing the BioLith team on their social media platform.
  However, this can only happen once the Ashesi Communication Department grants approval
  to the team.

![](https://static.igem.wiki/teams/4746/wiki/notebook/picture1.jpg)

![](https://static.igem.wiki/teams/4746/wiki/notebook/picture3.jpg)

###### Meeting with Cultivarium

#### WEEK 2 - JULY 10TH TO 14TH

* All the sub-teams within BioLith provided updates on
  their progress. The team kept on coming up with ideas
  for Human Practice activities. The laboratory team created Chloramphenicol LB agar
  plates and used four chromoproteins from the iGEM
  distribution kit, which were tsPurple, amaiLime,
  gfasPurple, and asPink, to transform bacteria.

![](https://static.igem.wiki/teams/4746/wiki/notebook/picture5.jpg)

###### Martha, one of the lab team members, making the Chloramphenicol LB agar plates

* BioLith team shared their descriptions to be featured on the social
  media platform of Integrated DNA Technologies (IDT).
  The Hardware team focused on designing the PCB and routing
  mechanisms. They also created a wireframe of the component
  layout to ensure a portable and compact design.

* Meanwhile, the website team addressed feedback by resolving
  issues on the website. The team had a tutorial session about
  parts and sequences with Dr. Rosca, and their advisor, Prince.

![](https://static.igem.wiki/teams/4746/wiki/notebook/picture6.jpg)

![](https://static.igem.wiki/teams/4746/wiki/notebook/picture7.jpg)

###### Tutorial session with Dr Rosca and Prince

* The Hardware team explored the idea of utilizing electromagnetism
  to insert and retrieve the device from the ground. Additionally,
  they shared the PCB design with a former Ashesi Ghana iGEM member
  for review.

* The team made sure that the gene sequences were accurate and
  recorded the various parts for effortless identification for
  future purposes. The website team began working on the Parts
  page of the website. The laboratory team began preparing
  the overnight culture.

#### WEEK 3 - JULY 17TH TO 21ST

* The website team received feedback and resolved issues with the Parts page.
  The team settled on a date to meet with another iGEM team, Penn State for
  collaboration purposes. The laboratory team proceeded with the overnight culture while the Hardware
  team utilized Fusion 360 to create a case for the components that are set
  to be placed on the PCB they recently constructed.

![](https://static.igem.wiki/teams/4746/wiki/notebook/picture8.jpg)

###### Lab team members working on overnight culture

* During the progress update meeting,
  the various sub-teams within BioLith
  shared their latest developments.
  The hardware team suggested creating an
  app to complement the project's design.
  BioLith also had a meeting with the Penn State iGEM team to explore possible
  collaboration opportunities.

* In addition, BioLith sought advice and feedback on their project
  from Ginkgo Bioworks, who are a group of mentors specialized in
  supporting iGEM teams throughout the competition.

![](https://static.igem.wiki/teams/4746/wiki/notebook/picture11.png)

###### Meeting session with the Penn State iGEM team.

![](https://static.igem.wiki/teams/4746/wiki/notebook/picture9.png)

###### Meeting session with Ginkgo Bioworks

#### WEEK 4 - JULY 24TH TO 31ST

* The website team completed the Atrributions page of the website. Entrepreneurship team made updates to the stakeholder and competitor's analyses. The first drafted script of the promotional video was completed. The wiki team started working on a wireframe for the Human practice page of the website.

* The lab-team had a first attempt at making hydrogels with guidance from the teams PI DR Rosca, and subsequently, made hydrogels of different concentration to determine which concentration would favor more bacteria growth and would also be visible for image processing by the modelling/software team.

![](https://static.igem.wiki/teams/4746/wiki/notebook/picture13.jpg)

###### First attempt at making hydrogels with Dr Rosca.

## AUGUST

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August

#### MAIN FOCUS OF AUGUST: DESIGN

#### HIGHLIGHTS OF THE MONTH

#### We transitioned from recording the notebook weekly to Monthly.

* The lab team conducted experiments on Hydrogels
  to determine the concentration of sodium alginate,
  the substance used to make the hydrogels. This will
  help ensure the highest efficiency of bacteria activity
  in detecting lithium in the ground. The results of these
  experiments led to changes in the hardware designed to
  encapsulate the hydrogels.

* We recently had the chance to present our project to the directors
  of Engineers and Planners Co Ltd with the hopes of collaborating
  with them in the future. During the meeting, we received valuable
  feedback regarding the placement of the biosensor and its impact
  on detecting lithium and associated elements. Additionally, they
  provided suggestions on how to enhance the retrieval process and
  prevent the holes from collapsing after inserting the biosensor into the ground.

![](https://static.igem.wiki/teams/4746/wiki/human-practice/human-practice/e-p-4.jpg)

![](https://static.igem.wiki/teams/4746/wiki/human-practice/e-p-2.jpg)

###### Meeting session with Engineers & Planners directors.

## SEPTEMBER

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September

####

#### HIGHLIGHTS OF THE MONTH:

* Wiki team created the Sustainable development page and made some edits to the Human Practices page.

* Software and Modelling team updated some functionalities on the software such as the synthetic biology learning page with a chat bot as well as order screen and payment type.

* Lab team performed tests to prove that the bacteria would be alive after five days and also begun hydrogel tests for GFB (green fluorescent protein).

## OCTOBER

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October

####

####

#### HIGHLIGHTS OF THE MONTH:

* The hardware team commenced a diffusion experiment to determine how long it takes the lithium and arsenic ions into 1% agarose by using dye. Furthermore, the team performed a mechanical testing on agarose which was a trial test to determine how easily it could break considering that it would be placed in the ground and could potentially experience some kind of loading.

![](https://static.igem.wiki/teams/4746/wiki/diffusion-1.png)

![](https://static.igem.wiki/teams/4746/wiki/diffusion-2.png)

###### Diffusion tests on the Agarose

* The team submitted the team booth safety form to get approval on bringing our built hardware to our booth at the grand Jamboree. Info session with Ashesi University students to inform them more about synthetic biology, share our project with them and encourage them to enroll in synthetic biology elective if interested. And some team members journeyed to East Airport International School to have a synthetic biology info session.

* Lab team performed a mini prep, made another batch of hydrogels for testing and also checked hydrogels for fluorescence. Entrepreneurship team made final touches to the business plan and interviewed students who stay in mining communities better understand the negative impact of mining on their lifestyle and environment as well as hear their any concerns they may have about our bacteria-based solution. The hardware team continued the mechanical testing, diffusion test and commenced their documentation and calculations. Electrical hardware team also begun making the PCB

![](https://static.igem.wiki/teams/4746/wiki/info-session-ashesi.png)

![](https://static.igem.wiki/teams/4746/wiki/info-session-east-airport.png)

###### Info sessions at Ashesi University and East Airport International School

* Software and Modelling team updated some functionalities on the software such as the synthetic biology learning page with a chat bot as well as order screen and payment type.

* Lab team performed tests to prove that the bacteria would be alive after five days and also begun hydrogel tests for GFB (green fluorescent protein). Furthermore, we had an interview with The Unit Manager for Exploration of Gold Fields to learn more about the current exploration and detection methods of gold and to pitch our project and receive some feedback.

![](https://static.igem.wiki/teams/4746/wiki/goldfield-2.png)

![](https://static.igem.wiki/teams/4746/wiki/goldfield-1.png)

###### Interview with the Unit Manager of Gold Field

## ENGINEEERING

![](https://static.igem.wiki/teams/4746/wiki/miner.jpg)

Welcome to the Engineering Page! Here, we take you on a captivating journey through the dynamic world of engineering, highlighting the intricate flow of ideas, the continuous iterations, both successes and failures, and the resilience it takes to develop groundbreaking solutions. Our projects were anything but straightforward, necessitating in-depth research, creative modeling, and persistent adjustments. We emphasize the pivotal role of iteration in engineering, where continuous refinement and learning from mistakes lead to progress. Alongside our achievements, we acknowledge and embrace failures as integral steps towards innovation. As you explore our engineering adventures, you'll witness the challenges we tackled, the cutting-edge research we harnessed, and the tools and technologies that fueled our endeavors. Join us in celebrating the engineering spirit that drives us forward, showcasing the boundless potential of this exciting field.

[Biological Engineering Cycle](#lab1)
[Mechanical Hardware cycle](#mech)
[Electrical Hardware cycle](#elec)
[Software cycle](#soft)
[Entrepreneurship cycle](#ent1)

### BIOLOGICAL ENGINEERING CYCLE

The engineering cycle is the foundation of our strategy in our iGEM project. From choosing our topic of interest to lab work, we employed the engineering design thinking process. We used an organized, iterative approach that promoted creativity and improvement for our bioengineered system. Here we describe the failures, successes and iterations made during our project.

![]( https://static.igem.wiki/teams/4746/wiki/edp.png)

The first steps involved topic selection followed by choosing a suitable strain of bacteria for Lithium and Arsenic detection and carefully creating the genetic circuit that powers our biosensor. This was followed by the hard calibration phase, where we made sure that the different components of our biosensor worked appropriately. By putting the various components of our biosensor through several testing, we were able to confirm its operation and therefore prepare it for practical applications. Every component of our sensor was chosen with safety and ethics in mind, highlighting ethical and responsible genetic engineering techniques. Our project's engineering flow drives continuous improvement and adaptation, ensuring our project remains on the cutting edge of synthetic biology and offers solutions to pressing challenges.

### TOPIC SELECTION

To begin with, we identified various problems within our community and ways in which we could help mitigate those problems. At this stage, intensive research was done on possible problems we could solve. After several literature review, we chose Lithium detection and mining using synthetic biology as Lithium is one of the promising industries. Looking at the product cycle of the lithium batteries, we realized these batteries end up in dumpsites after use creating another problem, environmental pollution. In addition, Lithium has just been discovered in some parts of Ghana and we intend to jump ahead to provide cheaper and sustainable means of detecting, mining and recycling lithium to prevent further damages to our environment. This influenced our decision to not only tackle Lithium detection and mining but also Lithium recycling.

### GENETIC CIRCUIT DESIGNS

Chromoproteins and fluorescent proteins as Reporters

In our project, we have strategically employed chromoproteins (CPs) as reporter genes to convert a transcriptional module into a quantifiable signal. Unlike fluorescent proteins (FPs), CPs offer several distinct advantages that align with our project's objectives. One notable advantage is the innate dark colour of CPs, making them easily discernible with the naked eye in ambient light conditions. This characteristic not only simplifies our detection process but also eliminates the need for costly and complex instrumentation, facilitating cost-effective examination. By opting for CPs as our reporter genes, we have leveraged their unique features to enhance the accessibility and practicality of our project.

Lithium sensing module

In a paper titled "Lithium-sensing riboswitch classes regulate expression of bacterial cation transporter genes" [1], the authors explored riboswitches, a genetic element, in sensing and regulating the expression of bacterial cation transporter genes related to lithium. By utilizing riboswitches, the researchers could detect the presence of lithium and subsequently control the expression of transporter genes responsible for moving cations, including lithium ions, across bacterial cell membranes. When the nhaA riboswitch detects the presence of Li+ ions it triggers the eforRed chromoprotein to be expressed thereby, indicating the presence of lithium.

Arsenic sensing module

By embracing the concept of employing an inducible promoter, we adopted, modified and improved the design from Ashesighana iGEM 2022 team, which hinged our design on a fascinating principle. This unique promoter remains in a dormant state until it encounters specific triggers, essentially waiting for an external stimulus to activate it. When the activator protein binds to the promoter in the presence of arsenite (As3+), it activates serving as an ignition for transcription. As a result, the reporter gene, amilGFP, emits a signal in the form of fluorescence (glow under blue light). This elegant mechanism offers a dynamic and controllable means of monitoring and reporting gene expression in response to arsenite exposure, a pivotal feature of our project.

![](https://static.igem.wiki/teams/4746/wiki/asdad.gif)

Schematic of the As sending module based on the inducible promoter

Why Arsenic?

In our pursuit of a reliable and efficient method for detecting lithium, we turn to arsenic as an ideal pathfinder element. Arsenic, a well-established pathfinder for various geochemical indicators, plays a crucial role in hinting at the presence of valuable elements, including lithium. An international team of researchers found that certain geological settings with elevated arsenic concentrations often coincide with lithium deposits. This relationship is akin to the link between arsenic and gold in mineralization, where arsenic enrichment in soil minerals facilitates the binding of gold. In a similar manner, arsenic-rich geological environments appear to host elevated lithium levels, making arsenic an informative indicator for lithium prospecting. Furthermore, soil samples taken from regions with known lithium deposits frequently reveal significant arsenic levels, solidifying arsenic's status as a valuable pathfinder element for lithium. feature of our project.

Bioleaching Module

The bioleaching module utilizes acid thiobacillus ferroxidants in oxidizing iron and sulphur containing compounds. It contains tetrathionate hydrolase enzyme which catalyses tetrathionate hydrolysis. This involves sulphur oxidation to generate sulphate, elemental sulphur and thiosulfate. This module also contains high potential iron sulphur (HiPIP) and a pH resistant gene. The HiPIP has a high redox rate effective in catalysing the oxidation reaction taking place. It oxidises ferrous iron to ferric iron which further oxidises the metal sulphides in the compound thereby speeding the mineral dissolution rate to obtain lithium. Through these processes, the Lithium ions are liberated out from the compound and leached out. During the bioleaching process, hydrogen ion concentration increases thereby lowering pH hence the need for pH resistant gene.

### DESIGN ITERATIONS

![](https://static.igem.wiki/teams/4746/wiki/whatsapp-image-2023-10-11-at-15-35-24.jpeg)

After modelling our modules in Benchling, we sent them to IDT for synthesis. They were able to make the arsenic module, but the HiPIP and Lithium had problems. The Li and HiPIP modules failed because the sequences contained a feature that created a hairpin in the terminator, which prompted us to modify the terminator using different types of terminators from the iGEM registry. Another reason contributing to the failure was a higher GC content.
To solve these issues, we did codon optimization to reduce the high GC content in the sequence and changed the double terminator to a single terminator. We resubmitted them for sequencing again. This time, they were able to make the lithium part, but the HiPIP could still not be made. Although they were able to synthesise the lithium part, there was a secondary product present in the synthesis as an impurity and as such required thorough screening for downstream application.

### HYDROGELS

Sodium Alginate Concentrations

For our hydrogels, we used 2.5%, 5% and 10% of sodium alginate with 5% Calcium chloride to experiment and establish an ideal sodium alginate concentration. The 2.5% hydrogels did not form the balls as desired but rather looked like a gel. The 10% sodium alginate hydrogels formed well defined spheres. Thus, through trying out different sodium alginate concentrations we were able to determine the ideal sodium alginate concentration as 10%.

Optical Densities

Initially we started making hydrogels with bacteria at OD600 of 0.01, 0.1 and 1.00. The colour change for hydrogel balls with 0.01 OD was not clearly visible whereas that of 1.0 was more visible. In an attempt to increase visibility, we performed increased the OD and experimented with 2.00 OD, 3.00 OD and 4.00 OD. An increase in OD showed an increased intensity in the colour. However, in as much as the colour was well pronounced, it did not change over time which meant that higher OD was not ideal for our case. Having realized this, we reduced the OD to maximum of 1.5 and minimum of 0.75. This was the OD range we proceeded to use for all our subsequent experiments.

### REFERENCES

[1] White, N. H., Sadeeshkumar, H., Sun, A., Sudarsan, N., & Breaker, R. R. (2022). Lithium-sensing riboswitch classes regulate expression of bacterial cation transporter genes. Scientific Reports, 12(1). https://doi.org/10.1038/s41598-022-20695-6

### BIOLITH ENTREPRENEURSHIP ENGINEERING CYCLE

The Entrepreneurship Subteam is an integral part of our project, and its primary goal is to ensure the project's alignment with market demands and to foster innovation in sustainable lithium mining practices. Our journey began with feedback from key stakeholders, including Engineers and Planners, and local communities. This feedback was pivotal in reshaping our project objectives and technical approach. As a result, we shifted our focus towards pre-exploratory technology for lithium mining and exploring the recycling of lithium from battery waste using genetically engineered bacteria.

### PROBLEM IDENTIFICATION & STAKEHOLDER FEEDBACK

We recognized the importance of incorporating stakeholder feedback into our project. Engineers and Planners advised us that our project is best suited for pre-exploratory technology rather than direct extraction, given the technical requirements involved. This insight guided our project's adjustment towards more exploratory objectives while maintaining our sustainability commitment.

![]( https://static.igem.wiki/teams/4746/wiki/find.jpg)

### REASSESSMENT OF PROJECT GOALS & OBJECTIVES

We revisited our project goals and made necessary adjustments in response to stakeholder feedback. We realigned our focus from extraction to pre-exploratory technology, ensuring we met the industry's needs and environmental standards.

### RESEARCH & DEVELOPMENT

After restructuring the entrepreneurship team to accommodate the changes, in parallel with the wet-lab subteam, we conducted comprehensive research focusing on pre-exploratory technology and the technical prerequisites for successful implementation. Additionally, we began exploring the use of genetically engineered bacteria for efficient lithium recycling from battery waste, and we discovered papers that had reports of how bioleaching can be used to achieve that.

![]( https://static.igem.wiki/teams/4746/wiki/group2.jpg)

Fig 1. Electrical Hardware timeline showing progress of our journey.

### TECHNOLOGY DEVELOPMENT & PROTOTYPING

The Entrepreneurship Subteam worked closely with other subteams to develop prototypes for pre-exploratory technology. These prototypes aimed to facilitate better lithium detection and sustainable mining practices. We also initiated testing and refinement of bacterial processes for lithium recycling.

### MARKET ANLYSIS & VALIDATION

We conducted thorough market research to ensure market alignment identifying potential customers and partners. Validation of the demand for pre-exploratory technology in the lithium mining industry shaped our project direction.

![](https://static.igem.wiki/teams/4746/wiki/marketanalysis.jpg)

### BUSINESS MODEL DEVELOPMENT

Our team formulated a robust business model tailored to the pre-exploratory technology. We defined revenue streams, outlined potential collaborations with recycling partners, and developed a sustainable financial plan.

### MONITORING & EVALUATION

We are committed to continuously monitoring project performance, customer feedback, and market dynamics. This data-driven approach ensures we adapt and improve as we progress, staying true to our sustainability goals.

### STAKEHOLDER ENGAGEMENT

We established ongoing communication with our stakeholders, including Engineers, Planners, and local communities. Transparency and dialogue were vital in ensuring all parties were informed and aligned with our project adjustments and progress.

### FEEDBACK LOOP

Maintaining an open and dynamic feedback loop with all stakeholders is a cornerstone of our approach. We consider ongoing feedback invaluable in refining our technology and project evolution. The project remains adaptable to changing market dynamics and emerging opportunities.

### SCALING & MARKET ENTRY

Upon successful piloting, we are now focused on scaling the deployment of our pre-exploratory technology in the lithium mining sector. Collaborations with mining companies and recycling facilities are being explored as we gear up for market entry.

![](https://static.igem.wiki/teams/4746/wiki/scale.jpg)

### CONCLUSION

The Entrepreneurship section of the BIOLITH team remains dedicated to the success of the project, continuously evolving and improving the approaches that align with market needs and industry standards. Our journey showcases our commitment to responsible and sustainable lithium mining practices, driven by feedback, innovation, and collaboration.

### BIOLITH ELECTRICAL HARDWARE ENGINEERING CYCLE

It is impossible to make mention of the biosensor without the electrical hardware as it forms a key part of our entire project. This hardware bridges the gap between the mechanical hardware and the software and modelling. It is composed of a camera we call the LithoCam, a device built by the team which is able to take images of the bar code on the lid of each biosensor and associate that bar code with its respective GPS location. This aids in uniquely identifying each biosensor when performing further analysis after retrieval from the ground. On our engineering path, several changes had to be made based on research and the desired functionalities of the device, but it allowed us to explore more components and broaden our knowledge scope regarding electronics. Each challenge was a major learning opportunity for us and a stepping stone for greater and better improvements on our design. Thus, we never admitted defeat when the going got tough and out of that struggle came forth our functioning and innovative LithoCam.

![](https://static.igem.wiki/teams/4746/wiki/criceng.jpg)

### DESIGN CYCLE FOR ELECTRICAL HARDWARE

![](https://static.igem.wiki/teams/4746/wiki/multicolor-illustrated-design-process-timeline-infographic-presentation.png)

Fig 1. Electrical Hardware timeline showing progress of our journey.

### STAGE 1

In our project development process, we initially identified the ESP32 CAM microcontroller as the most suitable choice based on a Pugh chart evaluation. However, due to its unavailability, we had to procure one, and as a preliminary step, we sought to demonstrate the feasibility of associating photographs taken with a camera to GPS coordinates using a microcontroller. This led us to utilize the Raspberry Pi, equipped with the Pixi 2 camera, as a proof of concept. The core project concept involved employing electrical hardware to capture images of hydrogel samples before and after their placement in the ground, followed by an analysis to map out the likelihood of finding lithium in specific areas. This concept included incorporating barcodes, linking them to GPS coordinates, saving data in a database, and visualizing sample collection locations on a map. To achieve this, barcode labels would be affixed to the device housing, which took the form of a cuboid with hydrogel samples flanking the biosensor. Given the remote nature of potential mining sites without internet access, we planned to store captured images on an SD card for subsequent processing at a location with internet connectivity.

![](https://static.igem.wiki/teams/4746/wiki/picture6.png)

Fig 2. Pugh Chart comparing the different microcontrollers.

![](https://static.igem.wiki/teams/4746/wiki/picture11.jpg)

Fig 3. GPS locations from neo6mv2 GPS module showing on the terminal of the Raspberry pi

![](https://static.igem.wiki/teams/4746/wiki/picture12.jpg)

Fig 4. A picture taken by the pixi 2 cam showing image saved GPS location

### STAGE 2

In implementing the esp32 cam we considered implementing the following:

* Push button – To manually take the pictures.
* OLED screen – To display the GPS coordinates where the picture was taken, and
  indicate when the camera has captured the image.
* Wireless power source – To power the device remotely so as to not require a plug-in-wall power supply.
* Code the GPS module – This had to be done to retrieve latitude and longitude readings from the module.

### CHANGES AND MODIFICATION

* The reset button on the esp32 cam was used as the button for taking pictures.
* Using an OLED screen required extra pins which the esp32 cam did not have. As a result, we considered two alternatives.

-Choice 1:

This circuit captures the picture after pressing the reset button. There will be a flash of light that indicates a picture being taken. The file name will have the GPS location of the place the picture was taken, as well as a number indicating the order of sequence of pictures. All these components are powered wirelessly using the Li-ion batteries, also the camera is coded to ‘shutdown’, if it is not in use. It wakes up only when the reset is pressed hence saving battery power as well.

-Choice 2: :

Since the ESP32 cam is a board that sacrifices so much of its pins for internal connections. when using both the camera and the microSD card there are so many internal connections. Unfortunately, the few pins left are suitable for use when including an OLED screen. A second microcontroller (ESP32 wroom) will therefore be used. This would mean we have slightly increased our hardware space, as we will have: ESP32 cam, ESP32, OLED, and a GPS module. This option perfectly displays GPS locations on the OLED and captures pictures and saves it on the SD card and it also tells when the picture is captured. Both microcontrollers communicate with each other using an interrupt pin.

![](https://static.igem.wiki/teams/4746/wiki/picture8.png)

![](https://static.igem.wiki/teams/4746/wiki/picture7.png)

Fig 5. Image of electrical setup with OLED screen and SD card file directory showing images taken and saved.

### SCHEMATIC PCB DESIGN

We considered the following:

* Reset button on ESP32 would have to serve as camera-capture button.
* It will have header pins.
* The SD-card will be able to be removed and inserted at will.
* GPS module has its antenna attached so we need a space where that will rest.
* The camera would have to be protruding outward.

![](https://static.igem.wiki/teams/4746/wiki/picture13.jpg)

Fig 6. Schematic diagram of camera

![](https://static.igem.wiki/teams/4746/wiki/capture.png)

Fig 7. PCB design of camera

### IDEA 2 CONCLUSION

The first idea is building an electrical device that can take a picture and save the picture on an SD Card using the GPS location as the name of the picture file. The plan was to use an ESP32 CAM to take pictures and a NEO-6MV2 GPS Module to take notes of the GPS locations. An initial electrical design was done using the above-mentioned electrical components as well as two 3.7v lithium-ion batteries and an LM2596 to step down the combined voltage from the batteries to 5V in order to supply power to the whole system.
After the pictures with their GPS location have been saved on the SD Card, the SD card will be placed in another device for processing and mapping out the information.

### STAGE 3

Following certain loopholes, we identified in our final design in stage 2, we had to make some changes. The loopholes and possible suggestions:

1. A screen to preview the object before a picture is taken was not considered. Following this, research was done, and it was discovered that a TFT screen can be used as the preview screen (the 3.5” inch TFT Touch Shield LCD Module was what was easily available and hence was used).

### IMPLEMENTING A TFT SCREEN

Including the TFT screen required the addition of another microcontroller because the esp32 cam did not have enough I/O pins to communicate with the 3.5” inch TFT Touch Shield LCD Module (communicates using 8-bit parallel pins) so an ESP32 DOIT DEVKIT was chosen. The idea was to allow the esp32 cam communicate the images it views serially or via local Wi-Fi network (using the Esp Now Protocol) to the ESP32 DOIT DEVKIT which give the TFT screen the necessary data to display the images.

Schematic of circuit with TFT screen

![](https://static.igem.wiki/teams/4746/wiki/picture9.png)

Fig 8. Schematic diagram with TFT screen

### STAGE 4

While exploring the implementation of the TFT screen, we had the opportunity to pitch our project to the fabrication laboratory expert on our campus. He brought to our attention that mobile phones now attribute pictures to locations once the user has their location turned on. He suggested we explore the idea of using an application to do all that we want our electrical hardware. Following this, we decided to analyse both ideas based on their pros and cons.

### ELECTRICAL HARDWARE SOLUTION:

Cost Analysis of Electrical Hardware Implementation

![](https://static.igem.wiki/teams/4746/wiki/capture2.png)

Fig 9. Table of cost analysis of camera

### PROs OF ELECTRICAL HARDWARE:

* 1. Easy to develop the final product.

### CONs OF ELECTRICAL HARDWARE:

* 1. A lot of processes and gadgets so information may be lost along the way.
* 2. Lower picture quality of two mega pixels.
* 3. Less seamless. More steps to undertake.
* 4. Costly
* 5. May look like a classroom project considering the tools and components being used.

### WEB APPLICATION SOLUTION:

This solution considers that our users may already have a smart phone and even if they do not have a smart phone, the cheapest smart phone is not as expensive as the total cost of making Solution 1. The web application solution considers that smart phones already can take pictures and take notes of GPS locations. Hence, an application could be created to:

* 1. Take pictures and GPS location.
* 2. Perform the processing of the pictures.
* 3. Mapping out the information.

### PROs:

* 1. Seamless for user
* 2. A cheap smart phone can cost as low as ₵200.
* 3. May look more professional.
* 4. Few processes
* 5. Better picture quality with cheap smart phones having a picture quality of 5-8 mega pixels.

### CONs OF ELECTRICAL HARDWARE:

* 1. More difficult to build (may take more time to build).

### STAGE 5

After conversations about our business model with our instructor and some research done by talking to other experts, we divided the market of our product to two groups. Huge progressive mining industries and conservative local miners. Progressive mining industries already use software that enhance their mining experience and may see the value in our software as a product we are selling to them. However, conservative local miners will want to see something physical if we want to sell our project as a product to them. Due to this, we decided to continue with the electrical hardware idea and the application idea to serve different customer bases. However, we added another application that would be coupled with the electrical hardware. If we take an area for prospecting, we will want the biosensors placed in the ground to be certain distances apart in that area. This second application directs the user to places where they can create holes in that specific area to insert the biosensor into the ground while following the distance apart rule.

### ELECTRICAL HARDWARE IN STAGE 5:

In stage 3, it was mentioned that we added the 3.5” inch TFT Touch Shield LCD Module to preview the images our camera views. However, we faced challenges sharing the images taken by the esp32 cam to the esp32 DOIT DEVKIT. This is because the images taken by the esp32 cam are saved in .jpg which can have sizes between hundred kilobytes or megabytes. However, if we are transmitting data from the esp32 cam to the esp32 DOIT DEVKIT serially or via ESP-NOW, the amount of data that can be sent at a time is 250 bytes. Hence, to transmit, the image data must be broken down and sent at intervals. Due to this:

1. The ESP32 kept crashing since we wanted to view the images in real-time and it was sending a lot of data within very short intervals.

2. Some of the image data got lost. Hence, image data at the TFT screen’s end was distorted.

This required us to find a preview screen that could be directly connected directly to the esp32 cam without all these issues. Hence, we need a TFT screen that required fewer pins to be used. We were able to finally acquire the Adafruit 2.8-inch TFT Touch shield which has the option to communicate via SPI. This meant we needed only five pins from the esp32 cam to directly connect the TFT screen to it with an additional two pins to supply power to the screen. We still needed the ESP32 DOIT DEVKIT because after the TFT screen was connected to the esp32 CAM, we needed UART pins and I/O pins for the connection of the GPS module and a button to take pictures. Below is the schematic of the final electrical design and PCB layout.

![](https://static.igem.wiki/teams/4746/wiki/picture10.png)

Fig 10. Schematic of final electrical design and PCB layout

![](https://static.igem.wiki/teams/4746/wiki/capture333.png)

Fig 11. 2D of front and back layer of PCB

![](https://static.igem.wiki/teams/4746/wiki/capture44.png)

Fig 12. 3D of front and back layer of PCB

Due to the orientation of the final frame of our electrical hardware and the fact that we are using through hole components, we had to create another board such that our esp32 cam and battery are on one side of the board and the rest of the components on the other side.

![](https://static.igem.wiki/teams/4746/wiki/capture434.png)

Fig 13. 2D and 3D layout of PCB containing the esp32 cam

Now we have been introduced to our biosensor and the LithoCam into detail, but what then? How do we make sense of the colour change observed and how do we put to use the images taken by the LithoCam. This is where the software steps into the picture and makes its long-awaited entrance in its perfection. The software is vital to the success of this project because it puts all the pieces together and makes sense of all the data collected, completing the detection process. Similar to all the other cycles, the software engineering cycle also experienced a series of twists and turns. Our path to success was a struggle because of limited data, paid softwares, APIs (Application Programming Interfaces) which were difficult to program, just to name a few. Nonetheless, we continued to follow the engineering design cycle, never gave defeat the upper hand and from the fruit of our labour came a functioning mobile app and a successful mapping application.

### BIOLITH HARDWARE ENGINEERING CYCLE

Our entire solution starts with the mechanical hardware(biosensor) which performs the actual detection and acts as the lithium explorer.It contains the sodium alginate hydrogels which change colour depending on the prescence of lithium and arsenic ions in the dug ground. SolidWorks (a Computer Aided Design software) was used for modelling the structure of the biosensor which would be placed into the ground. The design of the sensor was subjected to different iterations based on tests and experiments performed in the lab, as well as the feedback received from some of our stakeholders and other users. We were sure to integrate majority of the feedback received or find some innovative way to address any concerns raised from our stakeholders. Observe our progress from the start to the very end, each design an improvement of the next and each setback a learning opportunity to grow and be open-minded in designing.

### DESIGN 1

Our first design consisted of a hexagonally shaped containment with bacteria embedded in flat hydrogels with the following features:

* Slides – the strains of bacteria synthesized for the detection of lithium and arsenic would be contained in sodium alginate hydogels and placed in slides which would fit into the containment.
* Hexagonal containment – the hexagonal shape allows for maximum exposure by increasing the contact surfaces of the bacteria for the diffusion of metal ions.

![]( https://static.igem.wiki/teams/4746/wiki/autodesk-fusion-360-education-license-2023-10-11-15-06-29.gif)

Fig 1 First CAD model

PROBLEM ENCOUNTERED

The wet lab team experienced some difficulty in making the hydrogels in rectangular flat shapes during their experiments and they also tended to deform easily, hence the need to brainstorm another way to create the hydogels and called for re-evaluation.

### DESIGN 2

After finding out that it would not be possible for the gels to be rectangular such that they fit into the slides, further testing was performed, and the small spherical shapes of the hydrogels were proven to be better and firmer. Thus, the design had to be modified to suit this new discovery.

![](https://static.igem.wiki/teams/4746/wiki/picture14.jpg)

Fig 2. Second SolidWorks CAD Model

NEW DESIGN

The new design included the following features:

* Long wells (holes) – several of these would be created in the agarose mold using plastic rods and the hydrogels would be placed in them.
* Agarose gel containment – agarose would be cast into a cylindrical shape and would serve as a means to hold the bacteria.
* Magnetic covering – similar to the initial idea however it would be attached to a screw-like object which would be joined to the bottom of the overall structue to hold it all in place.

PROBLEM ENCOUNTERED

Few hydrogels could fit into the structure, meaning a lower sensitivity for detection of lithium and arsenic ions. It also lacked a casing to serve as a covering over the agarose containment.

### DESIGN 3

This design was an improvement on the 2nd design. To increase the sensitivity of our biosensor, we increased the number of holes and also included a casing for our biosensor to protect the agarose from easily breaking due to any vigourous movements before it has been deployed. This biosensor would be placed at a depth of 3 metres in the ground. Given the heightened sensitivity of the bacteria, we presumed that this depth would be optimal for detecting traces of the lithium element and its associated pathfinder element, arsenic.

![](https://static.igem.wiki/teams/4746/wiki/capture200.png)

Fig 3. Biosensor without its casing

![](https://static.igem.wiki/teams/4746/wiki/capture555.png)

Fig 4. Biosensor casing with plastic rods within

PROBLEM ENCOUNTERED

No way to uniquely identify each biosensor so that we can know can associate each biosensor to its GPS location.

### DESIGN 4

To be able to uniquely identify each biosensor for further analysis by our software we placed a bar code on the lid of each biosensor for easy tracking and identification. Following a comprehensive discussion with a prominent prospecting company in Ghana, Engineers, and Planners, we received invaluable feedback indicating that a depth of 3 meters merely reached the topsoil layer, and consequently, no significant findings were likely in that range. This feedback prompted us to reevaluate our approach, leading us to reconsider the depth and modify it to 15 meters below the surface and incorporating this into our design.

![](https://static.igem.wiki/teams/4746/wiki/capture1000.png)

Fig 3. Biosensor without its casing

![](https://static.igem.wiki/teams/4746/wiki/cha.png)

Fig 4. Biosensor casing with plastic rods within

![](https://static.igem.wiki/teams/4746/wiki/cad-modelling-timeline.png)

### BIOLITH SOFTWARE ENGINEERING CYCLE

Now we have been introduced to our biosensor and the LithoCam into detail, but what then? How do we make sense of the colour change observed and how do we put to use the images taken by the LithoCam. This is where the software steps into the picture and makes its long-awaited entrance in its perfection. The software is vital to the success of this project because it puts all the pieces together and makes sense of all the data collected, completing the detection process. Similar to all the other cycles, the software engineering cycle also experienced a series of twists and turns. Our path to success was a struggle because of limited data, paid softwares, APIs (Application Programming Interfaces) which were difficult to program, just to name a few. Nonetheless, we continued to follow the engineering design cycle, never gave defeat the upper hand and from the fruit of our labour came a functioning mobile app and a successful mapping application.

DESIGN CYCLE FOR SOFTWARE

![](https://static.igem.wiki/teams/4746/wiki/picture11.png)

Fig 1. Design cycle of BIOLITH software

### STAGE 1:CHARTING NEW TERRITORIES

We studied the breakthrough work carried out by previous iGEM teams before starting our ground-breaking endeavor. Our objective was to find any pertinent earlier research, as well as areas where we could improve current tools and use them to solve our particular situation. The 2017 Ashesi University team, the 2019 University of Edinburgh team, the 2020 University of Newcastle team, and the 2020 University of Texas team are just a few of the famous teams whose work we looked at. All of these outstanding teams had difficulty developing biosensors to deal with environmental problems.
Similar to our current mission, these teams employed either Green Fluorescent Protein (GFP) or chromoproteins to signal the presence of the elements they were investigating. However, their results offered only binary outcomes: the element was either present or absent.
A remarkable breakthrough emerged from the 2022 Ashesi University team, who adopted a novel approach. Instead of limiting themselves to simple binary detection, they used pathfinders to ascertain the likelihood of an element's presence based on quantifying color changes.
Our mission took inspiration from these prior projects, steering our focus towards the detection of lithium and its associated pathfinder elements for pre-exploration. Our unique twist? We aimed to undertake a more comprehensive analysis that involved generating a surface plot illustrating regions and their respective probabilities of containing lithium.
In essence, we built upon the foundations laid by these pioneering teams, harnessing their collective wisdom to refine our strategy. Our deviation from the norm was utilising a machine learning model to recognize color changes and our development of a probability model and a mapping model, culminating in a detailed surface plot depicting the likelihood of lithium presence in various areas.

### STAGE 2:THE QUEST FOR BLUE

Our journey commenced with the exploration of the 'Shades of Blue' color palette. Initially, we set out to design a logistic regression algorithm tailored to discern various shades of blue. Our hypothesis was rooted in the belief that the intensity of biosensors correlated with the quantity of lithium in the soil. However, a pivotal realization swiftly altered our course: color intensity did not correspond to lithium quantity. Still, given our biosensors' high specificity, it merely served as a marker for lithium's presence.
This marked a turning point in our approach. Drawing inspiration from the AshesiGhana 2022 team, which had successfully used Pathfinder elements for prospecting gold, we decided to apply a similar principle to our project. Thus, our revised strategy aimed to enhance our ability to detect lithium's presence in various regions accurately.

![](https://static.igem.wiki/teams/4746/wiki/capture123.png)

Fig2: Image showing different shades of blue

### STAGE 3:BRIDGING THE SPECTRUM

As our project evolved, we introduced two additional pathfinder minerals into our scope, necessitating the development of a probability model. Our objective was to translate the meaning behind each color change of the hydrogels into a numerical probability value, facilitating the creation of a comprehensive map depicting the likelihood of lithium deposits.
To test the viability of this approach, we engineered a Python script capable of generating images with three distinct colors in varying proportions. A secondary script was devised to systematically analyze each pixel in these images, recognizing and counting the colors. In our testing system, blue denoted lithium detection, red signaled the presence of pathfinder element 1 (Arsenic), and yellow represented pathfinder element 2 (Rubidium). The probability formula was thus conceived: The probability formula was then formulated as follows:

P(Lithium) = (0.5 \* number of blue hydrogels + 0.25 \* number of red hydrogels + 0.25 \* number of yellow hydrogels) divided by the total number of hydrogels.

The assignment of probabilities was deliberate: lithium received a 0.5 probability coefficient, while both pathfinder elements were assigned a probability of 0.25. This emphasized that detecting lithium alongside its pathfinder elements increased the likelihood of lithium's presence.
This approach enabled us to come up with a numerical value that is of more importance in generating a surface plot.

![](https://static.igem.wiki/teams/4746/wiki/picture122.png)

Fig 3: Snippet of code showing the generate\_random\_picture function used to generate the random images.

### STAGE 4:PLOTTING THE PROBABILITIES

In the fourth stage of our endeavour, we harnessed the probabilities derived from images to craft a comprehensive surface plot. This plot employed GPS coordinates on the X and Y axes to pinpoint specific locations, while the Z axis conveyed the calculated probabilities. The software we developed facilitated a visual representation of probability distribution across our area of interest, enhancing our capacity to identify regions with varying lithium concentrations.

### STAGE 5:ADAPTING TO REALITY

In the final phase of our project, we encountered constraints that compelled us to adapt our approach. We streamlined our focus due to limitations in available genetic components, opting to work exclusively with arsenic as the pathfinder alongside lithium. This shift in focus presented an opportunity to fine-tune our probability model:

![](https://static.igem.wiki/teams/4746/wiki/picture135.png)

R is the number of red hydrogels

Y is the number of yellow hydrogels

N is the total number of hydrogels.

P is probability

At this juncture, we deeply understood the specific colors our biosensors displayed upon detecting these minerals. Yellow signified arsenic, while red indicated lithium's presence.In tandem with these adjustments, we optimized the physical structure of our hydrogels, transitioning from a rectangular shape to a spherical form. This alteration enhanced the biosensors' interaction with the environment, improving their efficacy.
In response to these adjustments, we developed two key software components to support our project. The first was a K-Nearest Neighbor color detection software, designed to identify the specific color exhibited by sodium alginate hydrogels containing the bioindicators. This software played a pivotal role in the accurate classification of hydrogel color changes, streamlining the detection process.
The second software component was a mapping application, allowing us to plot and visualize the probabilities of finding lithium across different GPS locations. This mapping software offered a practical and informative means of displaying the spatial distribution of lithium probabilities, thus facilitating a more comprehensive understanding of the field of interest. These software developments were instrumental in optimizing our lithium and arsenic detection system.

Lastly, we introduced a mobile app designed to provide a seamless experience for our users. This app served multiple purposes:

* Facilitating communication with our team.
* Assisting with biosensor orders.
* Enabling users to view their generated surface plots.
* Aiding in the mapping of areas even in our absence.

Users could calculate the number of biosensors required for their exploration needs and utilize the BIOLITH camera to capture the barcodes of biosensors, correlating them with specific locations where the biosensors was placed. Below are the key features of the mobile app:

![](https://static.igem.wiki/teams/4746/wiki/capture4344.png)

![](https://static.igem.wiki/teams/4746/wiki/capture100.png)

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

### EXPERIMENTS

[Project Specific protocols](#prot)
[Viability Test on Hydrogels encapsulated bacteria](#via)
[Golden gate assembly protocol](#gate)
[Backbone Digestion](#back)
[Agarose Gel electrophoresis](#aga)
[General Protocol](#gen)

### 1. PROJECT SPECIFIC PROTOCOLS

Preparation of cells with Different Optical Densities (OD)

We used this protocol to prepare the different OD600 cultures of bacteria that we were going to encapsulate in the hydrogels. We carried out this experiment to determine which OD of bacteria culture will be ideal for observing color change in the hydrogels. First, we decided to prepare bacteria cultures which contained our desired plasmid (aeBlue) of ODs of 1.0, 0.1 and 0.01. We repeated this same experiment with bacteria that contained pJUMP24-1A(sfGFP) to obtain cultures with ODs of 1.5, 1.0 and 0.75.

1. We prepared a 1:100 overnight culture of the desired plasmids (aeBlue) contained in the NEB 10β E. coli bacteria strain in LB media.
2. The initial Optical Density of the overnight culture was measured to be 2.03 OD600. To obtain an OD600 of 1.0, 10 mls of the 2.03 OD600 culture was diluted in a 1:2 ratio by adding 10mls of LB media to get an OD of approximately 1.0.
3. Further dilution of the 1.0 OD600 culture was done in the ratio 1: 10 to get a cell culture with an OD600 of 0.1. This was achieved by transferring 2mls of the 1.0 OD600 culture to a falcon tube with 18 mls of LB media.
4. Further dilution of the 0.1 OD600 was done in the ration of 1: 10 to achieve a cell culture with an OD600 of 0.01. This was achieved by transferring 2mls of the 0.1 OD600 culture to a falcon tube with 18 mls of LB media. At the end of each dilution, the final culture was vortexed to properly mix.
5. To harvest the cells, we aliquoted 3mls of each cell culture into 3 different tubes (making 9 tubes in total – the 3 tubes for each OD culture were used in the next step for Preparation of sodium alginate hydrogel encapsulated bacteria for 3 different sodium alginate concentrations)
6. We centrifuged the cultures in the tubes at 4000 rpm for 10 minutes at 4 degrees Celsius temperature and then discarded the supernatant.

Note: The dilutions were achieved by using the equation: C1V1 = C2V2, where:

C1 = stock concentration, V1 = stock volume, C2= desired concentration, V2 = desired volume

![](https://static.igem.wiki/teams/4746/wiki/la.png)

Preparation of sodium alginate hydrogel encapsulated bacteria

This protocol was used to prepare the hydrogels. Sodium alginate hydrogels were prepared with Calcium Chloride as a cross linker. The Ca2+ from the solution interacts with the Sodium Alginate to create a three-dimensional gel structure. This allowed us to mold or create the desired hydrogel shapes.

1. 25 g of hydrated calcium chloride was dissolved in 500 ml (about 16.91 oz) distilled water in a beaker. This gave a 5% calcium chloride solution.
2. Different concentrations of sodium alginate were prepared using the steps outlined below:

* 2g of sodium alginate was dissolved in 20 ml (about 0.68 oz) of LB media to create a 10% alginate solution. The solution was vortexed slightly to mix.
* The 10% solution is dissolved in a ratio of 1:2 to get a solution of 5% by transferring 10 ml of the 10% Sodium Alginate solution to a falcon tube of 10 ml LB media. The solution was vortexed slightly to mix.
* The 5% Sodium Alginate solution was diluted in a ratio of 1:2 to get a solution of 2.5% by transferring 10 ml of the 5% solution to a falcon tube of 10 ml lb. The solution was vortexed slightly to mix.

The dilutions were achieved by using the equation: C1V1 = C2V2, where:

C1 = stock concentration, V1 = stock volume, C2= desired concentration, V2 = desired volume

3. For each of the tubes containing harvested cells from the cultures with different ODs from the **Preparation of cells with Different Optical Densities**, we resuspended the cells in 3mls of the different concentrations of sodium alginate. For example, the harvested cells from the bacteria culture of OD of 1.0 in three tubes, were resuspending in 3ml of concentrations of 10%, 5% and 2.5% sodium alginate respectively. This was repeated for the other tubes containing harvested cells from cultures with OD of 0.1 and 0.01. Below is a table with the description of the nine test categories.

![](https://static.igem.wiki/teams/4746/wiki/hj.png)

Table 1 Test categories for hydrogel encapsulated bacteria containing aeBlue plasmid

4. After the resuspensions, a pipette or dropper was used to make spherical droplets of the various solutions in the 5% Calcium Chloride concentration. Nine different containers containing the 5% Calcium Chloride solutions were labelled according to the different concentrations of Sodium Alginate and the OD of the bacteria that was encapsulated by the Sodium alginate.

5. The hydrogels were left in the Calcium Chloride solution for 15 minutes to fully crosslink.

6. The hydrogel balls were then transferred to petri dishes lined with wet paper towels (wet paper towels with distilled water to keep the hydrogels moisturised) and placed in an incubator at a temperature of 37 degrees Celsius to monitor bacterial growth by observing at specific time intervals, colour changes in the hydrogel.

![](https://static.igem.wiki/teams/4746/wiki/asd.png)

![](https://static.igem.wiki/teams/4746/wiki/fsa.png)

### Viability Test on Hydrogels encapsulated bacteria.

This test was done to check on the viability of bacteria in hydrogels after being allowed to grow in hydrogels over a period of 3 days. To determine the number of days that the bacteria can survive in the hydrogels, we used the following protocol.

1. One to three hydrogel spheres from the different test categories (cells at a particular OD resuspended in a particular concentration of Sodium Alginate) were placed in 5mls of LB media with antibiotic in different falcon tubes. There are nine different test categories as seen in Table 1.

2. Cultures were incubated with shaking at 37 °C and 140 rpm overnight.

3. The results were checked the next day. Cloudy cultures signify the growth of bacteria, indicating viable cells and vice versa. The O.D of the culture can be checked as well.

4. To clearly see the color, the overnight culture was centrifuged at 4°C for 10 minutes at 11,000 rpm. The supernatant was discarded leaving a pellet of cells left behind to also detect color changes.

![](https://static.igem.wiki/teams/4746/wiki/dgs.png)

### GOLDEN GATE ASSEMBLY PROTOCOL

After receiving our synthesized genetic parts, we used this protocol to assemble them.

1. We resuspended the Lithium Inducer, Arsenic Inducer and UV\_T4 endolysin DNA part to a concentration of 10ng/µl each.

2. The reagents below were added to two tubes, one containing the Li-sensor and another the As-sensor DNA parts.

![](https://static.igem.wiki/teams/4746/wiki/rea.png)

3. PCR was carried out at 37 °C 20 minutes, 37 °C 20 minutes 16°C 3 minutes for 15 times, 50°C 5 minutes, 80°C 5 minutes.

4. Transformation of the Li inducer plasmid and As inducer plasmid was carried out using the freshly prepared competent cells, BL21 and NEB 10 beta cells. Transformation was done following the general transformation protocol. Move to step 6.

5. Transformation was done again using the NEB5alpha commercial competent cells. The commercially competent cell protocol was used.

a. For each assembly, thaw a 50 µl tube of NEB 5-alpha competent cell on ice for 10 minutes.

b. Add 25 µl of the assembly reaction; gently mix by flicking the tube 4-5 times.

c. Incubate on ice for 30 min.

d. Heat shock at 42°C for 30 sec.

e. Place back on ice for 5 min.

f. Add 950 µl of room temperature SOC media. Incubate at 37°C for 60 min @250 r.p.m.

6. Plating was done with LB agar containing Kanamycin antibiotic.

7. Plated cells were incubated at 37 °C overnight.

8. Cell growths were inspected the next day under blue light and with the naked eye.

![](https://static.igem.wiki/teams/4746/wiki/ry.png)

### GOLDEN GATE ASSEMBLY PROTOCOL

This is another golden gate assembly method that we used to corroborate our results.

![](https://static.igem.wiki/teams/4746/wiki/xcv.png)

![](https://static.igem.wiki/teams/4746/wiki/pom.png)

1. The 25ul assembly reactions were set up as seen in the illustration above.

2. The reaction was mixed gently by pipetting up and down 4 times and transferred to the thermocycler and programed as follows: (5 min 37°C → 5 min 16°C) x 30 cycles followed by 5 min 60°C

3. Transformation was done using the NEB5alpha commercial competent cells. The commercially competent cell protocol was used.

a. For each assembly, thaw a 50 µl tube of NEB 5-alpha competent cell on ice for 10 minutes

b. Add 25 µl of the assembly reaction; gently mix by flicking the tube 4-5 times.

c. Incubate on ice for 30 min.

d. Heat shock at 42°C for 30 sec.

e. Place back on ice for 5 min.

4. 950 µl of room temperature SOC media was added and the mixture was incubated at 37°C for 60 min @250 rpm

5. LB agar plates containing kanamycin (for Pjump24-1A(sfGFP)) were warmed at 37°C for 15 min.

6. After the cells were mixed thoroughly by flicking the tube and inverting, 100 µl of the culture was spread onto each plate.

7. The plates were incubated overnight at 37°C.

### Backbone Digestion

pJUMP24-1A(sfGFP) digestion with Bsal-HFv2

This protocol was used to trouble shoot the results we got from the parts assembly. Seeing that the results we got were not as desired. We used several different BsaI-HFv2 enzymes for this digestion to ensure the plasmids were being cut to the right sizes. We also perform an insilico digestion of the pJUMP24-1A(sfGFP) using Benchling.

![](https://static.igem.wiki/teams/4746/wiki/nk.png)

1. Set up reaction as follows per 50ul reaction.

i. Add 8.7ul of 114.5ng/ml pJUMP24-1A(sfGFP)

ii. Add 5ul of 10X rCutSmart buffer.

iii. Add 1ul of Bsal-HFv2

iv. Add 35.3ul of nuclease free water.

2. Carry out PCR at 37°C for 20 mins and 60°C for 20 sec.

3. After PCR, conduct Gel Electrophoresis.

### AGAROSE GEL ELECTROPHORESIS

This experiment was used to confirm if we had the right parts in the plasmids.

Preparation

1. Add 80 ml of 1X TAE buffer into a glass laboratory bottle.

2. Add 0.8g agarose powder to the TAE buffer to make 1% agarose.

i) Calculated using this equation.

Agarose (grams) = Gel desired (%) x Volume 1x TBE buffer (mL)

3. Microwave until the agarose is completely dissolved.

i) (Do not overboil the solution, as some of the buffer will evaporate and thus alter the final percentage of agarose in the gel.)

ii) \* Be careful stirring, eruptive boiling can occur.

4. Let the agarose solution cool to 60 or70 degrees Celsius. Or until you can comfortably put your hand on the flask. \* Make sure it does not solidify!

5. Add 5uL of SafeView™ Classic or SafeView™ DNA Gel stain to the cooled agarose solution.

6. Pour the agarose into a gel tray with the well comb in place.

i) Pour slowly to avoid bubbles which will disrupt the gel.

ii) Make sure that the comb is nearest to the black electrode (cathode), as the DNA migrates towards the red electrode (anode).

7. Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified.

8. Once solidified, place the agarose gel into the gel box (electrophoresis unit).

9. Fill gel box with 1xTAE (or TBE) until the gel is covered.

10. Carefully load your samples into the additional wells of the gel.

11. Run the gel at 120V for 30minutes.

12. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.

13. Use a transilluminator or any device that has UV light to visualize the results.

### Loading Samples and Running an Agarose Gel

1. Add loading dye to each of your DNA samples. DNA samples for each well should be about 10 - 100ng. This protocol uses NEB 6x purple loading dye. To calculate the amount of loading buffer needed, use 1ul of 6x loading dye: 5ul DNA sample. Loading buffer serves two purposes:

i. It provides a visible dye that helps with gel loading and allows you to gauge how far the DNA has migrated.

ii. It contains a high percentage of glycerol that increases the density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer.

iii. a. Using the DNA ladder in the first lane as a guide (the manufacturer's instruction will tell you the size of each band), you can infer the size of the DNA in your sample lanes.

![](https://static.igem.wiki/teams/4746/wiki/pm.png)

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### Measurements & Results

| Measurement Questions | Path to section that answers question |
| --- | --- |
| Could the measurement(s) be repeated by other iGEM teams? | [Project Specific protocol](https://2023.igem.wiki/ashesighana/experiments#prot) |
| Is the protocol well described? | [Project Specific and General protocols](https://2023.igem.wiki/ashesighana/experiments) |
| Is it useful to other projects? | [Lab Contributions](https://2023.igem.wiki/ashesighana/contribution) |
| Did the team appropriately use controls to validate the measurement process and calibrate units? | [Parts Assembly](#assembly) [GEL Electrophoresis [Viability Test](#viability)](#gel) |

### OVERVIEW

This is a summary of the lab work we have been able to complete since we began working on our iGEM project and the iterations we used to improve results. We first describe the in-silico design process for the plasmids using the Benchling online software. We then describe the assembly process between the parts and the plasmid, the difficulties we encountered, and the troubleshooting we have done. We also outline the steps taken to enclose the bacteria in hydrogels as well as the outcomes of these experiments. An important part of the experiment was determining how long the bacteria could survive in the hydrogel, so we took the time to study the dynamics of cell viability in relation to the properties of the hydrogel.

[HiPIP & pH Resistance sequence &Tetrathionate Hydrolase](#hip)
[Bioleaching Plasmid](#bio)
[GEL Electrophoresis](#gel)
[Hydrogel synthesis](#hyd)

### In Silico Assembly of Genetic Parts Using Benchling

After preliminary research, we identified the important parts needed to create a lithium detector, its pathfinder element, Arsenic, and the genetic parts required for bioleaching. We moved on to designing the plasmid in silico using Benchling. The plasmids we designed contained two assembly standards: the Biobrick and Golden gate assembly methods. All the DNA sequences we designed contained a Biobrick prefix and suffix and a BsaI recognition sequence. When the BsaI restriction enzyme digests the genetic sequence, a 4bp overhang remains at the 5’ end of the DNA sequence, see Figure 1.

![](https://static.igem.wiki/teams/4746/wiki/dna-sequence.png)

Figure 1: DNA sequence skeleton

A successful assembly is influenced by the level of ligation fidelity of the overhangs. Our choice of the overhang base pairs was guided by a paper by Potatov et al [1]. The combination of base pairs we chose for ligation had a ligation fidelity of 99%. Additionally, having dual assembly standards allows us to have the flexibility to switch between multiple assemblies. This design also allows us to troubleshoot our assemblies if experiments do not yield the desired outcome. Furthermore, our selection of the two assembly standards was influenced by the pJUMP24-1A(sfGFP) backbone we used that contained both assembly standards. We used the pJUMP24-1A(sfGFP) because it contains a superfolded GFP that would allow us to screen colonies after assembly and transformation.

### Lithium Detector

After preliminary research, we identified the important parts needed to create a lithium detector, its pathfinder element, Arsenic, and the genetic parts required for bioleaching. We moved on to designing the plasmid in silico using Benchling. The plasmids we designed contained two assembly standards: the Biobrick and Golden gate assembly methods. All the DNA sequences we designed contained a Biobrick prefix and suffix and a BsaI recognition sequence. When the BsaI restriction enzyme digests the genetic sequence, a 4bp overhang remains at the 5’ end of the DNA sequence, see Figure 1.

![](https://static.igem.wiki/teams/4746/wiki/asdasdd.jpg)

Figure 2: Lithium sensor DNA sequence

The most significant part of our designs was the lithium sensing module. The module contains a BsaI cut site, 4 base pairs, and a bio brick prefix. The promoter initiates the biological circuit. The nhAa-I used for the sensing module was attained from a paper by White et al [2], which outlines the nhAa-I riboswitch could be used to detect the presence of Lithium ions. Once the nhAa-I senses the presence of lithium. The ribosome binding site binds to the ribosome and allows for the translation and expression of the chromoprotein eforRed indicating the presence of Lithium Ions. The terminator is important in stopping the biological circuit.

### Arsenic Detector

![](https://static.igem.wiki/teams/4746/wiki/sensor-ars.jpg)

Figure 3: Arsenic sensor DNA sequence

The 2022 Ashesi\_Ghana team identified Arsenic as a pathfinder element for Gold. Balaram & Sawant also identified Arsenic as a pathfinder element for most Lithium containing elements such as Lepidolite and Spodumene [3]. Detecting the presence of Arsenic in a specific place increases the probability of finding lithium and increases the probability of the E. coli bacteria correctly detecting the presence of lithium. The DNA sequence contains an arsenic sensing module that triggers the expression of the fluorescent protein AmilGFP that glows in the presence of blue light to show the presence of Arsenic.

### UV kill Switch

![](https://static.igem.wiki/teams/4746/wiki/uvasdad.jpg)

Figure 4: UV kill switch DNA sequence

This DNA sequence consists of the two assembly standard sequences, the UV promoter, ribosome, T4 endolysin and terminator. This creates a sequence that triggers the expression of T4 endolysin which causes the bacteria to self-destruct when exposed to UV light. This sequence is essential as it serves as a safety measure to prevent the bacteria from escaping into the environment.

### HiPIP & pH Resistance sequence &Tetrathionate Hydrolase

![](https://static.igem.wiki/teams/4746/wiki/hipipasdd.jpg)

Figure 5: HiPIP & pH resistance

This sequence provides a very significant element to the bioleaching module. It contains the HiPIP with a constitutive promoter, RBS and terminator. This part works together with the pH resistance gene to facilitate the reduction and oxidation of ions during bioleaching and at the same time allow the E. coli bacteria to survive in the harsh bioleaching pHs.

### HiPIP & pH Resistance sequence &Tetrathionate Hydrolase

![](https://static.igem.wiki/teams/4746/wiki/hipipasdd.jpg)

Figure 5: HiPIP & pH resistance

![](https://static.igem.wiki/teams/4746/wiki/tetraasdad.jpg)

Figure 6: Tetrathionate Hydrolase

### Benchling Digestion and Ligation of parts

The following plasmids were successfully digested and ligated in the Benchling software giving us a high probability that the assembly will be successful when carried out in the lab. Unfortunately, this did not prove to be correct for the bioleaching plasmid as it failed several times during synthesis.

### Lithium Sensing Module and Arsenic Sensing Module

The sensing modules were assembled using the benchling digestion and ligation assembly tool. This allowed us to select the parts to be assembled, followed by selecting the enzyme to use for cutting the parts. Since the overhangs created after digestion were compatible, benchling demonstrated that the parts are compatible, thereby leading to the assembled plasmid. The parts for the Lithium module were the lithium part, the UV\_T4 endolysin and the pJUMP24-1A(sfGFP) backbone. The Arsenic sensor on the other hand contained arsenic part, the UV\_T4 endolysin

![](https://static.igem.wiki/teams/4746/wiki/ligateasdsadsad.png)

Figure 7: Ligated Plasmid with Lithium Detector and UV Kill Switch

![](https://static.igem.wiki/teams/4746/wiki/ldsasdsadasdasd.png)

Figure 8: Ligated Plasmid with Arsenic Detector and UV Kill Switch

### Bioleaching Plasmid

The assembly of the bioleaching assembly contains the HiPIP, Hp Resistance gene, Tetrathionate Hydrolase and the pJUMP24-1A (sfGFP) backbone plasmid.

![](https://static.igem.wiki/teams/4746/wiki/asdsadsapodkaosd.png)

Figure 9: Ligated Plasmid with Arsenic Detector and UV Kill Switch

### In silico Digestion of pJUMP24-1A(sfGFP)

In order to verify that we were using the correct vector backbone in the reaction, we used benchling to perform an in-silico digestion using the BsaI restriction enzyme. From the digestion, the expected size of the uncut pJUMP24-1A (sfGFP) was 4399bp. As shown the virtual digest, the expected band sizes of the digested plasmid were 3560 bp and 839 bp respectively.

![](https://static.igem.wiki/teams/4746/wiki/aspdaciasdjna.png)

Figure G1: Expected results from Gel electrophoresis on Benchling

### Parts Assembly

Once the parts arrived from IDT (October 5th despite being ordered on July 17th) we prepared the stock concentrations of 10ng/µl of the Lithium and Arsenic and ran a golden gate assembly reaction. We then transformed the assembled plasmids (Lithium Sensing module and Arsenic Sensing Module) into three E. coli strands: BL21, NEB-10Beta, and NEB-5alpha using the Golden gate assembly protocol. Inspection of the different plates after incubation was done using the naked eye and under blue light. The results for the homemade competent cells (BL21, NEB10Beta). The results are as follows:

![](https://static.igem.wiki/teams/4746/wiki/measurement-1.png)

![](https://static.igem.wiki/teams/4746/wiki/measurement-2.png)

![](https://static.igem.wiki/teams/4746/wiki/measurement-3.png)

The plates with the BL21 cells had no growth indicating a problem with the cells. However, there was visible growth in the NEB 10-beta cells. A yellow color could be observed with the naked eye, indicating the presence of GFP. Since the plasmid contained a GFP gen, we also viewed the bacterial plates under UV light. The cells were glowing under UV light, indicating that the assembly did not work as the GFP was not knocked out through the assembly.

These are the results using a commercial competent cell NEB 5-alpha.

![](https://static.igem.wiki/teams/4746/wiki/measurement-4.png)

![](https://static.igem.wiki/teams/4746/wiki/measurement-5.png)

![](https://static.igem.wiki/teams/4746/wiki/measurement-6.png)

There was visible cell growth in the plates. The color was observable using the naked eye and under UV light.

Since the assembly was unsuccessful, we took a step back to troubleshoot. Our first troubleshooting step was to check if the BsaI-HFv2 enzyme used was working effectively. We ran a digestion with the old BSA1 and new BsaI-HFv2 enzymes and ran a Gel Electrophoresis.

### GEL Electrophoresis

We ran the digested pJUMP24-1A(sfGFP) on a 1% agarose gel and used NEB 1kb DNA ladder (Figure G3) to identify the inserts. The results of the Gel electrophoresis are shown below. Figure G1 shows the expected results from the Benchling simulation, and G2 shows the results from the lab experiment. Comparing G1 and G2, the ladder maker in G2 was between 3560 bp and (850bp), which corresponded to the fragments in G2, meaning the size was as expected, ndicating that Bsal was working correctly. The results showed that the new and old Bsal were cutting and working correctly. The new BsaI-HFv2 produced more intense bands than the old BSa1 enzyme indicating that it was more efficient at digesting DNA.

![](https://static.igem.wiki/teams/4746/wiki/gelsadsapda.png)

![](https://static.igem.wiki/teams/4746/wiki/sapdkapodksakckmc.png)

### Golden gate assembly using NEB protocol

We tried another golden gate assembly protocol by NEB to assemble the Lithium and Arsenic plasmids in hopes that the protocol would yield a different outcome. After the end of the experiment, the plates with Arsenic had bacteria colonies that glowed under UV light. However, the plates which contained the Lithium Sensing plasmid had a total of 11 colonies and 5 of them were not glowing under UV light. This indicates the colonies that were not glowing (approx.
50%) had possibly been carrying a successfully assembly. More tests are planned to prove this assumption.

![](https://static.igem.wiki/teams/4746/wiki/measurement-7.png)

![](https://static.igem.wiki/teams/4746/wiki/measurement-8.png)

![](https://static.igem.wiki/teams/4746/wiki/measurement-9.png)

### Hydrogel synthesis

#### Phase One Experiment

This part aimed to encapsulate E. coli bacteria in a Sodium Alginate hydrogel to create a safely contained and sensitive sensing module.
The first step was to determine the hydrogel concentrations with the best structural integrity and simultaneously allow for the growth of the bacteria. The experiment carried out was with three difference concentrations of bacteria (1.00, 0.1 OD, and 0.01 OD). These bacterial densities were resuspended in 3mls of different concentrations of Sodium Alginate (2.5%, 5%, and 10%) crosslinked with 5% Calcium Chloride.

![](https://static.igem.wiki/teams/4746/wiki/mjciss.png)

Figure R1:2.5%,5% & 10% sodium alginate solution

After placing the hydrogels in a petri dish and incubated at 37 °C. the plates were monitored for 5 days.

### Phase One Results

The 10% alginate hydrogels demonstrated the best structural integrity, while the 2.5% hydrogels had issues forming individual entities. Additionally, the OD of the cell culture (cell densities) influenced the extent of visible color changes in the hydrogel after the incubation period, with the OD of 1.0 resulting in the most significant change.

![](https://static.igem.wiki/teams/4746/wiki/xmcxozkczock.png)

### Implications of Phase One Results

The results suggest that a higher cell culture density (1.0 OD) expresses a deeper blue color change in the hydrogels, while lower densities (0.1 and 0.01 OD) do not. We recognized the need for higher cell concentrations to achieve a visible color change in the hydrogels. As a result, we conducted another experiment using cells with an optical density (O.D.) of 1.0 and above. The hydrogels with a 10% alginate concentration displayed the best structural integrity, and the cells within these hydrogels grew optimally. We utilized hydrogels from a 10% alginate concentration to ensure favorable cell growth and viability conditions.

### Phase Two Experiments

We proceeded to carry out another experiment with higher optical densities. We used aeblue cells ( E.coli contain colorimetric plasmid) with OD measurements of 4.0, 3.0, 2.0.

![](https://static.igem.wiki/teams/4746/wiki/mxzpcmz.png)

Figure R2: 2.0 O.D, 3.0 O.D & 4.0 O.D 10% sodium alginate solutions

### Phase Two Results

The color of the hydrogel with the cells with an optical density of 4.0 expressed a deeper blue color from the beginning, followed by the cell culture with an optical density of 3.0 and 2.0. After a day of incubation, the cells presented an even more pronounced blue color. The was no change in color from day two until day 5.

![](https://static.igem.wiki/teams/4746/wiki/odfmdcd.png)

We also noted that the cells' optical density (OD) appears to have a direct relationship with the expression of the blue colour in the hydrogels, which corroborated our earlier findings. The bacteria may have stopped expressing the blue colour due to a lack of nutrients or entering the lag phase of growth.

#### Phase Three Experiment

From these findings we moved on to using cells between the optical density of 0.1 and 1.0 ODs.
For our final experiment we used the Optical densities of 0.25, 0.50, 0.75 and 1.00 OD to find out which concentration would give us a stable cell growth and color change.

![](https://static.igem.wiki/teams/4746/wiki/mosadkaoskda.jpg)

Figure R2: 0.25 O.D, 0.5 O.D, 0.75 0.D & 1.0 O.D 10% sodium alginate solutions

### Phase Three Results

There was no noticeable color change in the hydrogels made. One assumption was that the cells were too old, so they were no longer expressing the blue color.

![](https://static.igem.wiki/teams/4746/wiki/mosxsaxmsaxpoaxax.png)

### Viability Test

Since the hydrogels had not expressed any blue color we ran a viability test on the hydrogels, to check if the cells were still growing and expressing the blue color. Below are the results we obtained, confirming that the cells were alive and growing.

![](https://static.igem.wiki/teams/4746/wiki/joijoihoioiho.jpg)

Figure R4: Overnight culture of 0.75 O.D, 0.5 0.D & 0.25 O.D cell solution.

Since the hydrogels had not expressed any blue color we ran a viability test on the hydrogels, to check if the cells were still growing and expressing the blue color. Below are the results we obtained, confirming that the cells were alive and growing.

![](https://static.igem.wiki/teams/4746/wiki/iuhuhoihidex.png)

The hydrogel beads did not turn blue as expected. However blue dots were observed on some of the beads.

![](https://static.igem.wiki/teams/4746/wiki/ndsajoax.jpg)

After the encapsulation, we assessed cellular viability. Below are the picture of the cells pre-shaking. Cells were grown before being centrifuged and collected for an overnight culture, producing a recognizable blue mass of cells at the bottom of the tube. Viability was assessed through naked-eye observation, optical density measurement, and color assessment of the collected group after centrifugation.

![](https://static.igem.wiki/teams/4746/wiki/jsoaimxsaxas.png)

Figure 11: Hydrogels in incushaker.

![](https://static.igem.wiki/teams/4746/wiki/gdfuiuhhuyrx.png)

Figure 11: Hydrogels in incushaker.

![](https://static.igem.wiki/teams/4746/wiki/dcacsc.png)

### Hydrogel Synthesis Using GFP

To further corroborate the results we repeated the experiment using hydrogels containing bacteria with a green florescent protein. This approach should have an increased sensitivity than the colorimetric approach.

![](https://static.igem.wiki/teams/4746/wiki/oasdjaoidnx.jpg)

Over a three-day observation period, we examined the Petri dishes and recorded measurements daily. Although the color change was less visible to the naked eye, we viewed the plates under UV light, revealing a notable fluorescence of the hydrogel spheres containing the cells.

### GFP plates under blue Light

![](https://static.igem.wiki/teams/4746/wiki/asjdnsaodmoakmd.png)

![](https://static.igem.wiki/teams/4746/wiki/aosiamksamdsaodsad.png)

### Conclusion

From our results, we successfully identified the optimal concentration required to create a hydrogel with strong structural integrity while simultaneously facilitating optimal bacterial growth. In the context of our project, it was crucial for the bacteria to remain within the hydrogel for a minimum of 5 days when deployed into the ground. To ascertain whether the cells would continue to grow and interact with the Lithium and Arsenic ions during this period, we conducted a viability test spanning 5 days. Our results demonstrated that the cells remained viable throughout this duration. To further validate our findings, we employed two different types of bacteria, namely aeblue and GFP, and successfully substantiated our claims. Although these experiments were not conducted with the inducible cells, one we create the inducible cells and characterize their induction and sensitivity we can directly substitute the required concentration of cells to create the biosensing unit.

[1] V. Potatov, J. Ong, R. Kucera, B. Longhorrst, and K. Billot, “Comprehensive profiling of four base overhang ... - ACS publications,” ACSPublications, https://pubs.acs.org/doi/10.1021/acssynbio.8b00333 (accessed Oct. 11, 2023).

[2] N. White, H. Sadeeshkumar, A. Sun, N. Sudarsan, and R. R. Breaker, “Lithium-sensing riboswitch classes regulate expression of bacterial cation transporter genes,” Scientific Reports, vol. 12, no. 1, 2022. doi:10.1038/s41598-022-20695-6

[3] V. Balaram and S. S. Sawant, “Indicator Minerals, Pathfinder elements, and portable analytical instruments in Mineral Exploration Studies,” MDPI, https://www.mdpi.com/2075-163X/12/4/394#:~:text=In%20the%20case%20of%20lithium,and%20spodumene%20(Table%201). (accessed Oct. 12, 2023).

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