iGEM 2022 — Team ashesighana

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## NOTEBOOK

>

## APRIL

-

Meetups to decide the project we are doing, which area in mining we were tackling and preparations to kick start the project.

## JUNE

+

### Week 1 – June 8th to June 10th

We have officially started our internship/competition!!
This was the week when the entire team came together! As a result, two other students from the Kwame Nkrumah University of Science and Technology (KNUST) with whom we collaborated, came to Ashesi University for a week to kick off the project.

![](https://static.igem.wiki/teams/4186/wiki/april-1-1.png)

### Day 1: 8th June 2022

We started the day excellently with two other team members in the AshesiGhana team outside our institution. They talked about what they do on campus, their interest and how excited they are about the project.

A brief introduction was followed by the Principal Instructor (PI), Dr Elena Rosca. We discussed what Synthetic biology is doing and how we can leverage it to solve world pressing problems, especially issues identified in the pre-exploratory stage of gold mining in Ghana.

We spent the afternoon brainstorming on the research and scoping the project topic. We were tasked with conducting additional research on biosensors, how gold can be explored, exploratory mining methods and principles, and so on. By the end of the day, we had clearly defined the project topic and identified some biological methods for detecting gold particles or ions.

### Day 2: 9th June 2022

We created a Slack page, Google Docs folder, and OneDrive folder to keep the team's files and important documents. In addition, different co-Leads/sub-teams were chosen for the various project responsibilities and components.

* Design - Vera, Sandra, and Betty
* Modelling - Gideon
* Wiki - Elijah
* Collaborations - Trish and Michael
* Human Practices - Edith and Micheal
* Documentation and Notebook - Vera

We also brainstormed and proposed human practices to ensure an integrated human practice.

### Day 3: 10th June 2022

We continued with a detailed suggestion of integrated human practices and education. The team investigated applying for the impact Grant; hence, we reviewed the questions. Our PI introduced us to Benching, a cloud-based platform for biotechnology and research. We explored some of the platform's features that would be useful to us in our project.

### Week 2 - June 13th to June 16th

### Day 1: 13th June 2022

The team completed the Impact grant application and requested that the PI review it. Michael, the team's co-lead, ensured this and chaired the application process by gathering relevant information and conducting a literature review for the application.

### Day 2: 14th June 2022

Further discussion on our project resulted in us coming up with a problem statement and solid literature to back our research idea and proposed solution. At this stage, we hadn't looked at the biological composition of our solution; we just explored the potential solutions without considering the biological feasibility.

From there, we looked at the first level of detection of gold complexes or ions, bacteria culture for the specific detection and other pathfinder elements. We also asked the question; how do we turn off these bacteria after sensing?

### Day 3: 15th June 2022

We looked into iron, arsenic, and gold binding proteins. We also looked at genetic circuits and inducible promoters for the different pathfinders. A resource person took the team through benchling on how to do sequencing, take notes, perform digestion, ligation, and cloning, among others.

### Day 4: 16th June 2022

To turn the gene, we looked into inducible promoters for the selected pathfinders and catalytic enzymes. Also, our two team members from the other institution finally left us for school! 😔😢

![](https://static.igem.wiki/teams/4186/wiki/april-3-1.png)

![](https://static.igem.wiki/teams/4186/wiki/april-2-1.png)

### Week 3 – June 20th to June 24th

### Day 1: 20th June 2022

The team brainstormed and researched solutions to the question, "How do we turn these bacteria off after sensing?" This session's ideas gave birth to our first safety and biosecurity stage, which includes using a UV kill switch to activate the T4 Endolysin. Given that we have a diverse team with little to no biology or SynBio background, two design team members designed their first sequence of the arsenic inducer in benchling (See figure below!). The rest of the team sat through benchling tutorials on bio-bricks, digestion and ligation, and golden gate assembly.

![](https://static.igem.wiki/teams/4186/wiki/image-1.png)

### Day 2-5: 21st – 25th June 2022

The team extensively discussed integrated human practices, education and collaborations with elaborate objectives and required actions. We also met with the Map the System (MTS) Ashesi Team, who also worked on "A systems analysis of the political, environmental, and socio-economic impacts of Illegal Mining in Ghana". Insights from this meeting helped us with a power dynamics 2x2 matrix, a stakeholder map and a clear list of other stakeholders we need to integrate into our project to create a user-centered solution.

![](https://static.igem.wiki/teams/4186/wiki/image-2.png)

Also, Gideon Bonsu and Vera Bordah migrated from benchling to snap gene to do the designs. The experience was a great one!

### Week 4 – June 27th to July 1st

### Day 2-5: 21st – 25th June 2022

Sub teams were assigned a task to investigate the safety and biosecurity grant application, specific secretion of proteins outside the selected chassis that enables it to interact with the gold and other pathfinder ions or complexes.

### Day 2: 28th June 2022

Leeroy explored other ways of safety besides the autolytic kill switch. This resulted in the 2nd and 3rd phases of our biosecurity and safety level. Thus, developing a hydrogel encapsulation that prevents bacteria from escaping into the environment and, secondly, methanol dependency for our chassis (E-coli strain).
The team researched arsenic, iron and gold ions binding proteins concerning our chassis.
Also, Stony Brook iGEM 2022 team reached out for collaboration on their outreach project.

### Day 3: 29th June 2022

Further research on these were made:

* why arsenic is an ideal element (pathfinder) to be detected
* Proposed alternative design for the arsenic sensing module inspired by Pengsong Li et al. research on the "development of a whole-cell biosensor based on an ArsR-Pars regulatory circuit."

![](https://static.igem.wiki/teams/4186/wiki/image-3.png)

![](https://static.igem.wiki/teams/4186/wiki/image-4.png)

## JULY

+

### Week 5 - July 4th to July 8th

### Day 1: 4th July 2022

The team explored to find the sequence for the arsenic promoter, thus Pars and arsR.

### Day 2: 5th July 2022

The team has a zoom call with our PI, Dr. Elena Rosca, via zoom to discuss our sequences, running them in benchling to enable us to submit our part to twist for sequencing. We also discussed a developing framework for documenting information from human practices: interviews, audio recordings, and write-ups, among others. Edith went ahead to contact Ghana Minerals Commission and Golden Star Mining Company. Lastly, Gideon helped the team with writing the project description on slack!

### Day 3: 6th July 2022

We researched why iron is an ideal element to be detected and the interesting relation between iron and our selected chassis (E-coli). From this research, we identified ferrous iron repressor FUR.
We also explored some promoters controlled by FUR protein. These were:

* AceB promoter
* Fes promoter
* FepA promoter
* yncE promoter

Also, we found out that “although the FUR acts as a transcriptional repressor it can be turned into an activator by preventing or eliminating another transcriptional repressor’s ability to bind to DNA. These findings resulted in our first iron sensing module….

Pc -> -> (RBS)->Fur -> (T) -> PAceB ->-> (RBS)-> lacI-> (T) -> PlacI -> eforRed->(T)

Also, we officially met with Stony Brook to discuss the terms of a possible partnership. They proposed that we participate in their journal initiative, where we describe the societal impact of our project. And we also proposed that they could help us with modelling.

![](https://static.igem.wiki/teams/4186/wiki/image-5.png)

### Day 4: 7th July 2022

The team decided to investigate alternative designs due to the issue of leakages in our first designs. Our initial design had limited control and operated in a direct "input-output” paradigm. To prevent false positives would lead to an ineffective biosensor that will further worsen the problem we seek to address by giving false indications of areas that may not have gold. Thus, strong terminators insulate against read-through of genes from neighboring promoters.

### Week 6 – July 11th to July 15th

### Day 1: 11th July 2022

Early Monday morning this week, Gideon and Vera worked on updating the iron sensing module so we could submit it to IDT and twist. The updated iron sensing module was:
Pc -> (RBS)->Fur -> (T) -> PAceB ->-> (RBS)-> TetR-> (T) -> pTetR ->RBS -> eforRed->(T)
Thus, we replaced the lacI repressor and its regulatory promoter with TetR and pTetR respectively.
In preparation for our meeting with the minerals commission of Ghana, as part of human practices, the team used this time to prepare for the meeting adequately. The co-lead of the team, Michael, helped with the slides.

![](https://static.igem.wiki/teams/4186/wiki/image-6.png)

### Day 2-3: 12th – 13th July 2022

We designed the kill switch gene sequence in snap gene, and all other designs that were submitted to IDT and Twist.
pUV ->RBS->T4 Endolysin->Terminator

![](https://static.igem.wiki/teams/4186/wiki/image-7.png)

![](https://static.igem.wiki/teams/4186/wiki/image-8.png)

![](https://static.igem.wiki/teams/4186/wiki/image-9.png)

![](https://static.igem.wiki/teams/4186/wiki/image-10.png)

### Day 4: 14th July 2022

Some team members, Gideon, Edith, Michael, and Trish, met with the Minerals Commission of Ghana to understand the current pre-exploratory gold mining activities, the consequences or the impact of the current approaches and any chance of using synthetic biology in this pre-exploratory stage. The team pitched our solution to them and got relevant feedback to improve it.

![](https://static.igem.wiki/teams/4186/wiki/image-11.png)

![](https://static.igem.wiki/teams/4186/wiki/image-12.png)

### Week 7 – July 18th to July 22nd

### Day 1: 18th July 2022

We got our safety training!
We also had a brainstorming session following our meeting with Ghana's minerals commission to discuss their feedback and how to incorporate it into our design. This birthed the staking idea. To develop the stake design, we first demonstrated with a tube filled with a slime where the slime represented the hydrogel we planned to design as a safety measure.

![](https://static.igem.wiki/teams/4186/wiki/image-13.png)

![](https://static.igem.wiki/teams/4186/wiki/image-14.png)

### Day 2: 19th July 2022

A day after the safety training, the team set up the lab space to kickstart lab work for the project.

![](https://static.igem.wiki/teams/4186/wiki/image-15.png)

![](https://static.igem.wiki/teams/4186/wiki/image-16.png)

### Day 3: 20th July 2022

We looked at the different lab equipment and what they do and performed simple lab activities such as pipetting, using the water bath, centrifuge etc. We also prepared LB Media and made plates with and without ampicillin before the transformation we were going to do.

![](https://static.igem.wiki/teams/4186/wiki/image-17.png)

![](https://static.igem.wiki/teams/4186/wiki/image-18.png)

### Day 4: 21st July 2022

Some team members were doing wet lab work, and others were developing content for the wiki page. The two co-leads for the team, Michael and Trish, and Gideon met with team Exeter to discuss a collaboration regarding examining their CAD model. We agreed to the collaboration and ask of them in return to troubleshoot our initial models after we complete them.

![](https://static.igem.wiki/teams/4186/wiki/image-19.png)

It was an exciting day for the team! Approximately 80% of us had no previous experience with synthetic biology. As a result, today's experiments in engineering bacteria strain were an exciting part of our journey. We formed the first competent cells! It was a fun moment, a learning experience, and the starting point for everything we needed to do in the lab to complete our project.

### Day 5:22nd July 2022

As part of education under human practices, the team had a session with some students in the United Student Achievers Program in Zimbabwe. These high schoolers were working on degrading plastic utilizing an engineered microorganism and wanted help with some information to aid them in achieving their goal.

### Week 7 – July 18th to July 22nd

### Day 1:25th July 2022

The team compiled all the parts we used for our design and provided the links to them on the iGEM registry to aid in efficient parts identification and their functions (the role they play in the sequence).

### Day 2:26th July 2022

We checked the protocol for transformation since it was the next thing for us to do. Also, we delegated tasks to populate the wiki content document. Edith was tasked to write on the project description and landing page, Vera with design, Gideon with modeling, Trish with collaborations, Leeroy with Safety, Rosemond with Teams page and Michael with human practices.

### Day 3:27th July 2022

Individuals continued to work on populating the wiki page content document. We also had a photoshoot for the wiki page, promo video and to thank Inquaba BioTech for providing us with some reagents, lab coats and other lab items. We finalized the designs of our genetic circuits and developed graphic representations of them in PowerPoint.

![](https://static.igem.wiki/teams/4186/wiki/image-20.png)

### Day 4:28th July 2022

We met with Stony Brook via zoom to further discuss the project's direction and the entities to model in the system. They suggested possible modeling approaches.

![](https://static.igem.wiki/teams/4186/wiki/image-21.png)

### Day 5:29th July 2022

The deadline for the promo video was approaching fast. Led by Michael, Elijah, and Gideon, we brainstormed ideas for the promo video. Gideon also developed models for reactions in the genetic circuits using the Simbiology library in MATLAB. He sent the models to the University of Exeter for their review and feedback, given our collaboration with them.

![](https://static.igem.wiki/teams/4186/wiki/image-22.png)

## AUGUST

+

### Week 9 – August 1st to 4th

### Day 1: August 1st, 2022

July was very busy, demanding and a step closer to the final jamboree. We started the Month with a high spirit. We were one of the teams to win the Impact Grant! How exciting is that?

![](https://static.igem.wiki/teams/4186/wiki/image-23.png)

In the first week of August, we made a transformation with amilGFP, amilCP an meffRED from the iGEM kits we got. Unfortunately, our ice marker got spoilt hence we had to make ice from frozen sachet water by crashing it with a mallet. That was a great way to resolve the problem with the shortest time.
We also had a meeting with Exeter to work on our collaboration.

### Day 2: August 2nd, 2022

Our transformation worked! Yaaay. We were excited to see our transformation work given that it was the first time for about 80% of us. With the help of our PI, we sent sponsorship letters to different organizations such as Tullow Ghana, AngloGold Ashanti etc.; we also went through the content we had written for the wiki page to edit and finalize some parts of it.
Later in the day, some team members met with an engineer from the exploratory division of Golden Star Mining Company. We presented our project to her and found out more about the exploration process and exactly where our solution would fit.

![](https://static.igem.wiki/teams/4186/wiki/image-24.png)

![](https://static.igem.wiki/teams/4186/wiki/image-25.png)

### Day 3: August 3rd, 2022

Each member helped to perform a mini prep using the zymoPURE miniprep kit. Also, videos of the team working in the lab were taken by the communications department from Ashesi university.

### Day 4: August 4th, 2022

We prepared another batch of competent cells. As part of human practices, we brainstormed ideas for synthetic biology info session at a high school called African Science Academy. Edith went on to arrange this session with the school.

### Day 5: August 6th, 2022

We had an info session with the Ashesi University community where we talked about synthetic biology and its application and encouraged students to enrol in the newly introduced synthetic biology course at the university.

![](https://static.igem.wiki/teams/4186/wiki/image-26.png)

![](https://static.igem.wiki/teams/4186/wiki/image-27.png)

### Week 10 – August 8th to 12th

Four of the team members went on a week-long break to take care of other essential engagements they had. However, Leeroy and Gideon helped to design and finalize a project on a biosensor for Stony Brook because we collaborated with them.

![](https://static.igem.wiki/teams/4186/wiki/image-28.png)

Also, the modeling team got feedback from Mat, university of Exeter.

### Week 11 – August 15th to 19th

This was a week before the start of the academic year; hence, team members were allowed to prepare for the academic year. We drew a detailed schedule showing each person busy and available to aid in planning to continue the project while doing academic work.
On Wednesday, August 17th, Gideon met with Mat to further discuss the feedback from his initial assessment and send over the designs of our genetic circuits for him to help us develop the elementary differential equations.
On Friday, August 19th, Mat from Stony Brook sent over the finished differential equations for gold and iron sensing models and walked us through how to represent them in Simbiology. Also, we received a google doc containing a modelling guide, differential equations and parameter values for our gold sensing module. This is very helpful!

![](https://static.igem.wiki/teams/4186/wiki/image-29.png)

Later in the day, we got some exciting news! We won the Safety and Biosecurity Grant.

![](https://static.igem.wiki/teams/4186/wiki/image-30.png)

### Week 12 – August 22nd to 26th

Our IDT and Twist parts arrived. We started by digesting and ligating the parts.
This was a tedious experience. We transformed the DNA after digestion and ligation, but there was no growth. That was devastating, but it was only the tip of the iceberg. Elijah does his best to populate the wiki page with as much information as possible before it freezes.

## SEPTEMTBER

+

### Week 13

The wiki guy lost his code…Shocking right? This week involved a lot of work in the wet lab and weekend work in the lab. Rosemond and Vera were busy troubleshooting the cause of the plasmid not ligating through serious digestion and ligation approaches. This included digesting pUC19 with two restriction enzymes, XbaI and SpeI. Michael was busy working on infographics and the content of our human practice, whiles Elijah was on the wiki.

### Week 14

From the results we obtained from our gel electrophoresis, we noticed that the pUC 19 wasn't showing. Hence, to rectify that, we made different concentrations of pUC 19 that we used to do different transformations. This was all we worked on for the entire week while other teammates were working on the wiki.

![](https://static.igem.wiki/teams/4186/wiki/image-31.png)

### Week 15

Finally, we had some light at the end of the tunnel, a different miniprep, and the gel result showed a plasmid.
Also, on the 13th of September, some team members met with engineers and planners (E & P). E & P are a leading indigenous Mining and Construction company in the West Africa sub-region. Their primary services include Mining, Construction & Civil Works, and Consulting Services. Following our presentation to their representatives, they provided inquiries, recommendations, and contributions

![](https://static.igem.wiki/teams/4186/wiki/image-32.png)

### Week 16

We started this week with a lot of excitement; with the new pUC 19 plasmids, we were able to get growth on the E Coli, and there was a bacteria glow; thus, green fluoresce since we had GFP reporter gene.

![](https://static.igem.wiki/teams/4186/wiki/image-33.png)

On 21st of September, which was a government holiday, as part of education and collaboration, the team had a fun day with synthetic biology at African Science Academy (A-level school in Ghana), where:

1. We introduced the students to synthetic biology and the amazing things being done in the field

2. Extracted some DNA from bananas

3. Created DNA strand with Haribo and gummy bears

4. Finally, we demonstrated and worked through how to design a plasmid using coloured beads and a locker to represent the different parts of the plasmid.

![](https://static.igem.wiki/teams/4186/wiki/image-34.png)

![](https://static.igem.wiki/teams/4186/wiki/image-35.png)

Also, to know the concentration of the pUC 19 used to obtain the successful results, Rosemond and Vera, with a supporting Lab assistant, performed gel Electrophoresis, which whored that the pUC 19 with a ratio of 1:10 was the best choice.

![](https://static.igem.wiki/teams/4186/wiki/image-36.png)

![](https://static.igem.wiki/teams/4186/wiki/image-37.png)

### Week 17

We conducted digestion, ligation and transformation, including control setups with the 1:10 Puc 19 part to troubleshoot if our ligation and digestion worked.
We also wanted to determine which digestion approach was the best for our experiment, whether the digestion approach using a mastermix or without a mastermix.
Troubleshooting was successful, which we confirmed from the growth on our amp plates and gel electrophoresis. We confirmed that the digestion without the master mix gave the desired results; hence, we proceeded with our experiment with that information.

![](https://static.igem.wiki/teams/4186/wiki/image-38.png)

![](https://static.igem.wiki/teams/4186/wiki/image-39.png)

## OCTOBER

+

### Week 18

Officially the month of wiki freeze! Wrapping Up!
We conducted our final digestion, ligation and transformation experiment with our genetic parts. We then continued with a miniprep and Polymerase Chain Reaction (PCR) to amplify our gene of interest in order to conduct a gel electrophoresis.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

## ENGINEERING

## OVERVIEW

Welcome to the engineering page!.
Here we describe the engineering flow, the iterations, the successes, and the failures, and how, as we developed, conducted in-depth research, created, and modelled, we encountered difficulties that required many iterations and adjustments. You will see in this section that our project's development was not straightforward, making it worthwhile to highlight.

## ITERATIVE DESIGN IN SCOPE

The design process to come up with our part sequence was an iterative one. During the design stage of our project, we explored and evaluated leading to a refinement in our designs and reworked in response to the problems that emerged as the analysis was conducted with our design.

![](https://static.igem.wiki/teams/4186/wiki/iterations-1.png)

## GENETIC CIRCUIT DESIGNS

🤔 Why Arsenic is an Ideal Element to be Detected.

An international team of researchers have found that when some soil minerals are enriched with arsenic (e.g. pyrite), gold can enter the mineral structural sites by directly binding to arsenic (forming, chemically speaking, Au(2+) and As(1-) bonds, which allows gold to be stabilized in the mineral. However, when the arsenic concentration is low, gold doesn't enter the mineral structure but only forms weak gold-sulfur bonds with the mineral surface [2]. This implies a relationship between the presence of arsenic and gold. Put another way, a patch of land with high arsenic concentrations is more likely to harbor short-term gold.

Per data collected in the Birimian of southwestern Ghana, where significant gold deposits have been found, arsenic concentrations of up to 40-58 ppm and Au concentrations of 200-260 ppm have been detected in prospective gold areas. In addition, it was found that pathfinder components suited for characterizing Au mineralization in intricate regolith settings were found to be As and Zn[3]. Soils overlying sulfide ore deposits commonly contain arsenic at several hundred parts per million; the reported maximum is 8,000 ppm. This arsenic may be present in unweathered sulfide minerals or an inorganic anion state [4].

Arsenic exists in the environment in two forms: organic and inorganic arsenic species. However, inorganic arsenic species, which include arsenite and arsenate, are the most prevalent forms of arsenic in the environment [5]. It should be noted that some of these sulfides, such as pyrite, with inorganic arsenic, come together to accumulate gold. From these findings and evidence, we can engineer the bacteria to detect arsenite (As (III), an inorganic form of arsenic).

🤔 Why Iron is an Ideal Element to be Detected.

Our initial design didn't include Iron as a pathfinder; however, to increase the chance of getting gold in the explored land using our biosensor, we discovered Iron as another potential pathfinder and included it in our design.

Instead of destroying E. coli, iron (Fe) causes two intriguing things to happen to it. Firstly, iron stimulates growth as it is an essential metabolite not available to E. coli in serum. Secondly, it interferes with bacterial killing, a process usually mediated by natural antibodies and complements [7]. However, the bacteria limit the iron pathogens can access during infection by generating iron- and siderophore-chelating proteins, exporting iron from intracellular compartments containing infections, and regulating dietary iron absorption. A transcription factor called Ferric Uptake Regulator (Fur) uses Fe2+ , as a corepressor. It inhibits the uptake of iron in pathogens [8].

In the presence of Ferrous ions, FUR acts as a repressor; In other words, it binds with the Fe2+ to undergo structural change. mRNA transcription is then inhibited when the homodimeric Fur- Fe2+ complex attaches to the DNA at a Fur binding site. However, when Fe2+ is absent, the mRNA is allowed to transcribe. Although FUR acts as a transcriptional repressor, it can be turned into an activator by preventing or eliminating another transcriptional repressor's ability to bind to DNA [8].

## INITIAL GOLD SENSING MODULE

This design utilized an inducible Au promoter triggered in the presence of gold ions (Au+, for transcription, eventually causing the eforRed reporter protein to express a pink colouration (figure 1).

![](https://static.igem.wiki/teams/4186/wiki/initial-gold-1.png)

Figure 1a: Initial schematic of the Au sensing module based on the inducible promoter

## INITIAL ARSENIC SENSING MODULE

With the idea of utilizing an inducible promoter, thus, the promoter comes on or becomes active in the cell in response to specific stimuli. They stay in an inactive stage unless they receive stimuli [11]. This served as the basis for our initial design (Fig. 2). The activator protein binds to the promoter in the presence of arsenite (As3+) and activates it to start transcription. This causes the reporter gene (amilGFP) to signal the transcriptional activity in the bacteria by causing the bacteria to glow.

![](https://static.igem.wiki/teams/4186/wiki/initial-gold-2.png)

Figure 2a: Initial schematic of the As sending module based on the inducible promoter

## Justification for The Need to Iterate The Initial Biosensing Designs

Biosensor systems have certain shortcomings, such as high leakage, low induced fold change and poor sensitivity. Leakages in genetic circuits exacerbate false positives. This is primarily true with linear designs. However, we need to use “secure” circuits that reduce leakages, hence, false positives. Typically, false positives would lead to an ineffective biosensor that will further worsen the problem we seek to address by giving false indications of areas that may not have gold [1]. Thus, this will increase pits and failed extractions. Our initial designs had limited control and operated in a direct "input-output” paradigm.

Also, in the initial designs, we utilized Au and As pathfinders. Although these trace elements were good pathfinders for gold prospecting, to increase the probability of the presence of gold after prospecting, we included another vital pathfinder for gold, thus, Iron (Fe) oxides. Studies in identifying pathfinder elements for gold in some areas of Ghana revealed that Fe is strongly associated with gold. Hence, the alternate and final designs incorporated the detection of Fe2+.

## Final Gold Sensing Module

![](https://static.igem.wiki/teams/4186/wiki/gols.gif)

Figure 1b: Schematic of the sensing module for Au based on the PgolTS-golS-PgolB-amilCP regulatory circuit in the presence of Au

## FINAL ARSENIC SENSING MODULE

![](https://static.igem.wiki/teams/4186/wiki/arsr.gif)

Figure 2b: Schematic of the sensing module for As based on the arsR operon which regulates As+

## FINAL IRON SENSING MODULE

![](https://static.igem.wiki/teams/4186/wiki/fur.gif)

Figure 3: Schematic of the sensing module for Fe based on the FUR-PAceB-TetR-Ptet module in the presence of Fe2+

## BIO-STAKE DESIGNS

The evolution of the development of the bio-stake design within which the hydrogels(more about this in the safety section) are placed is highlighted below:

![](https://static.igem.wiki/teams/4186/wiki/human-engineering.png)

Figure 4: Engineering Design Evolution

## REFERENCES

* [1] J. A. N. Brophy and c. A. Voigt, "Principles of genetic circuit design," Nature Methods, vol. 11, pp. 508-520, April 2014.
* [2] G. conference, "Goldfinder: scientists discover why we can find gold at all," 4 July 2021. [Online]. Available: https://www.eurekalert.org/news-releases/555387. [Accessed July 2022].
* [3] E. Arhin, S. Boadi and M. C. Esoah, "Identifying pathfinder elements from termite mound samples for gold exploration in regolith complex terrain of the Lawra belt, NW Ghana," Journal of African Earth Sciences, vol. 109, pp. 143-153, 2015.
* [4] D. o. E. a. L. Studies and C. o. L. Sciences, "Distribution of Arsenic in the Environment," in Arsenic: Medical and Biologic Effects of Environmental Pollutants., Washington DC, National Academies Press, 1977.
* [5] E. E. J. M. P. Eduardo Moreno-Jiménez, "The fate of arsenic in soil-plant systems," Reviews of Environmental Contamination and Toxicology, vol. 215, pp. 1-37, 2011.
* [6] S. Udayangani, "Difference Between.com," 2 January 2021. [Online]. Available: https://www.differencebetween.com/difference-between-inducible-and-constitutive-promoter/. [Accessed July 2022].
* [7] J. Fletcher, "The Effect of Iron and Transferrin on the Killing of Escherichia coli in Fresh Serum," Immunology, pp. 493-500, 1971
* [8] B. Troxell and H. M. Hassan, "Transcriptional regulation by Ferric Uptake Regulator (Fur) in pathogenic bacteria," Fronteirs in Cellular and Infectiob Microbiology, vol. 3, no. 59, 2013.

{% endblock %}

{% 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 %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## RESULTS

### OVERVIEW

The results outline the experimental work carried out over 1-2 months of lab work. First, we describe the construction and characterization of the various sensing modules cloned in the E-coli strain using
Snapgene. To separate the DNA fragments according to size and charge after a restriction digest, the team
simulated agarose gel electrophoresis in SnapGene. The second part presents the preliminary experiments
showing the digestion and ligation of our parts. With lots of research and iterations, the team designed
the genetic parts for our project in silico with the SnapGene application and sent them out for
synthetization. Our less experienced team with biological lab work (70% of the team are computer
science, mechanical and electrical engineering majors) started practicing in the lab with digestions,
ligations, transformations, gel electrophoresis, and making competent cells. We also explain the troubleshooting of these initial experiments
determining the issues with the ligation and digestion. Then, we explain the corrections of the digestion and ligation protocols resulting from this troubleshooting.
Also, we present the results obtained from the final try, continued with miniprep, PCR and gel electrophoresis to characterize the part. And finally, the result for the
first try of the hydrogel encapsulation and some proposed future experiments to obtain the expected results.

### PRELIMINARY SIMULATIONS

Before performing this experiment in the Lab, the team utilized Snapgene to simulate the digestion and ligation of the various parts. In this approach, we first performed a linear ligation between the sensing module and kill switch fragments (Au and UV, As and UV, Fe part 1 & 2 and UV) to evaluate if it's feasible to be ligated. This was done successfully, as represented in figures (1a,2a, and 3a). We used restriction and insertion cloning to insert these fragments into the plasmid (pUC 19). By inserting, we cut the plasmid with the EcoR1 and Pst1 to create sticky ends to facilitate ligation. We also cut the fragments with EcoR1 and PSt1 to maximize ligation efficiency. This resulted in complete plasmids with various sensing module inserts, as shown in figures 1b, 2b, and 3b. Figure 1c. 2c and 3c depict the vectors and inserted fragments with their various sizes (base pairs), which enabled the team to visualize how the insertion was done and the size of the various components.

1. Au and Kill Switch Model

![](https://static.igem.wiki/teams/4186/wiki/fig-1a.jpg)

Figure 1a: Linear ligation of gold inducer and the kill switch to create a complete gene insert

![](https://static.igem.wiki/teams/4186/wiki/fig-1b.jpg)

Figure 1b: Ligated plasmid with Au-UV insert

![](https://static.igem.wiki/teams/4186/wiki/fig-1c1.jpg)

![](https://static.igem.wiki/teams/4186/wiki/fig-1c2.jpg)

Figure 1c: Illustration depicting the vector (pUC 19) and the fragment (Au inducer-UV)

 2. As and Kill switch Module

![](https://static.igem.wiki/teams/4186/wiki/fig-2a.jpg)

Figure 2a: Linear ligation of arsenic inducer and the kill switch to create a complete gene insert

![](https://static.igem.wiki/teams/4186/wiki/fig-2b.jpg)

Figure 2b: Ligated plasmid with As-UV insert

![](https://static.igem.wiki/teams/4186/wiki/fig-2c1.jpg)

![](https://static.igem.wiki/teams/4186/wiki/fig-2c2.jpg)

Figure 2c: Illustration depicting the vector (pUC 19) and the fragment (As inducer-UV)

 3. Fe and Kill switch Module

![](https://static.igem.wiki/teams/4186/wiki/fig-3a.jpg)

Figure 3a: Linear ligation of ferric inducer and the kill switch to create a complete gene insert

![](https://static.igem.wiki/teams/4186/wiki/fig-3b.jpg)

Figure 3b: Ligated plasmid with As-UV insert

![](https://static.igem.wiki/teams/4186/wiki/fig-3c1.jpg)

![](https://static.igem.wiki/teams/4186/wiki/fig-3c2.jpg)

Figure 3c: Illustration depicting the vector (pUC 19) and the fragment (Ferric inducer-UV)

### GEL ELECTROPORESIS

In this simulation, the team wanted to identify the inserted fragments that bear the inducers or sensing modules. From the ligation and cloning, the expected sizes for Fe, Au and As-UV fragments were 2455bp, 2368bp and 2324bp, respectively. These sizes were checked with the 1% agarose gel electrophoresis, where the NEB 1 kb DNA ladder was used for the simulation to quantify the inserts and the DNAs. From figure 4, using the ladder marker at 3.0kb, we obtained the expected size as shown. From here, we concluded that since the simulation works, we could go ahead and do the lab experiments to confirm what is expected from the simulations.

![](https://static.igem.wiki/teams/4186/wiki/fig-4.jpg)

Figure 4: Identifying fragments that bear the inserted genes (Au, Fe and As inducers) by gel electrophoresis simulation.

### WET LAB EXPERIMENTS AND RESULTS

Once the parts arrived, we went ahead with digestion of our genetic parts or genes of interest (Gold [Au], Arsenic [As] and Iron [Fe (I & II)] sensing modules, and the autolytic UV kill switch modules) and the vector we chose for our design (Puc19 Plasmid). We continued with ligating the different gene fragments with the vector and performed transformation with the chassis we chose (Escherichia coli). While doing these experiments, we also made competent cells from bacteria culture that were grown overnight.

![](https://static.igem.wiki/teams/4186/wiki/fig-5.png)

Figure 5: Infographics showing the experimental processes that were undertaken

1.1 DIGESTION

We made 25 ul solutions for each digestion. We made three different tubes of digested puc19 DNA parts, three different tubes with digested Gold, Arsenic, and Iron (I), and three different tubes with two digested UV kill switch modules and an Fe (II) genetic part.
After these digestions were conducted, we went ahead with ligating the various corresponding parts with the digested vector.

![](https://static.igem.wiki/teams/4186/wiki/digestion.svg)

After these digestions were conducted, we went ahead with ligating the various corresponding parts with the digested vector.

1.2 LIGATION

We made 25 ul solutions for each digestion. We made three different tubes of digested puc19 DNA parts, three different tubes with digested Gold, Arsenic, and Iron (I), and three different tubes with two digested UV kill switch modules and an Fe (II) genetic part.
After these digestions were conducted, we went ahead with ligating the various corresponding parts with the digested vector.

![](https://static.igem.wiki/teams/4186/wiki/ligation.svg)

After ligation, we transformed the parts from the three tubes from ligation with the competent cells and allowed for overnight growth.

1.3 OBSERVATION, RESULTS AND TROUBLESHOOT

After the overnight growth, we did not observe any growth or colonies on our agar plates for Arsenic and Fe sensing modules. However, we observed very few colonies for the Au sensing module. We took the colony and added it to ampicillin media, added some gold particle-chippings and grew it overnight. We plated the culture with the gold particles on ampicillin plates to observe growth and color change over a period. No color change was observed for this investigation, however.
We then suspected something might have been wrong with the concentration of the plasmid used. We suspected the plasmid might have been over diluted to undergo digestion and ligation. Thus, we sought to find out if our suspicion was correct. We performed a digestion, ligation, and transformation for only the puc19 plasmid to obtain a very concentrated plasmid after we conducted a mini prep. After the mini prep, we ran a gel, to confirm if we were correct. From the gel image shown below we were correct. The second well which had the initial diluted plasmid DNA had no trace of DNA on its column, while the next four wells which had the concentrated plasmid had traces of DNA on their columns.

![](https://static.igem.wiki/teams/4186/wiki/fig-6.jpg)

Figure 6: Gel electrophoresis results comparing two different plasmids obtained with different miniprep kits to the NEB 1kb MW ladder

Another suspicion we had about the results we had was whether the mini-prep kit we used was problematic. Thus, for the gel image shown above, we conducted two mini-prep with an old and a new kit. The columns labelled A1 and A2 were minipreps conducted with the new kit and the columns labelled B1 and B2 were minipreps conducted with the old kit. We had two columns for each kit used because we used two different colonies for each kit. Based on the results from the gel, we concluded that both our old and new mini-prep kits had no issues.

1.4 PLASMID CONCENTRATION TROUBLE SHOOT

We were able to troubleshoot what was wrong and realized the plasmid DNA we used initially was very diluted. After obtaining the concentrated plasmid DNA, we went ahead to ascertain what concentration of the current plasmid DNA used would be ideal for our experiments. We diluted the current concentrated plasmid DNA into three concentrations (1:2 concentration, 1:5 concentration and a 1:10 concentration). We ran a gel and based on the results from the gel image below, we chose to go with a 1:10 concentration for our subsequent experiments.

![](https://static.igem.wiki/teams/4186/wiki/fig-7.jpg)

Figure 7: Gel electrophoresis results comparing different pUC 19 ratios

1.5 DIGESTION AND LIGATION TROUBLE SHOOT

The initial results obtained also raised concerns about whether we had issues with the digestion and ligation of our parts. Thus, before we went ahead with the second lab experiment with the remaining of our synthesized parts, we wanted to ascertain if our digestion and ligation worked with no errors. As a result, we performed digestion and ligation with the 1:10 concentrated plasmid DNA from the previous experiment. We digested with the restriction enzymes, SpeI and XbaI since both when used to cut the vector would have sticky ends that are compatible. For this experiment, we used two different approaches to digestion. One digestion approach involved making a Mastermix made up of the reagents used in digestion and the other approach involved no Mastermix. We suspected that making a Mastermix before digestion might further dilute the concentrations of the restriction enzymes and the DNA parts. Thus, we conducted these experiments to make sure our suspicions were correct or otherwise.

For this experiment, we had four digestions of pUC 19. Two of these digestions were done using the Mastermix approach (MM) and the other two were digested using the No Mastermix approach (No MM). One of the digested Puc19 part tubes for each digestion approach were transformed directly while the other two for each digestion approach went ahead to ligation.

We figured out that, if digestion worked then, the transformation for the digested parts from each approach should have no growth when plated on the ampicillin agar plates. The reasoning behind this is that, if the plasmids were not correctly cut or digested, the bacteria would have full plasmids with ampicillin resistance which would mean that these bacteria would not be killed by the ampicillin antibiotic. However, if they were cut correctly, the bacteria would not have ampicillin resistance from the plasmid DNA and would be destroyed by the ampicillin antibiotic from the agar plate and thus would experience no growth.

We further ligated the other two tubes, one each from the two digestion approaches, transformed with competent cells, and allowed for growth overnight. We had two control experimental setups for this experiment:

➕ Positive control (transformed E. coli bacteria with undigested and unligated Puc19 plasmid)

➖ Negative control (transformed E. coli bacteria only)

The positive control is expected to have growth because the E. coli bacteria would have ampicillin resistance from the plasmid DNA and would be destroyed by the ampicillin antibiotic from the agar plates. The negative control however is expected not to have any growth since it has no plasmid with ampicillin resistance and will be destroyed by the ampicillin antibiotic from the agar plate.

![](https://static.igem.wiki/teams/4186/wiki/fig-8.png)

Figure 8: results obtained from Digestion and ligation troubleshoot (a) transformed digested plasmid with master mix (b) transformed digested plasmid without master mix (straight NEB protocol) (c) Transformed ligated plasmid with master mix (d)Transformed ligated plasmid with no master mix (e) positive control (pUC 19 + competent cells) (f) Negative control (Just competent cells with no plasmid or DNA)

From the results we obtained after transformation as seen above, we had growth for the following experimental setups:

- Transformation using direct digested Puc19 from the Mastermix D(MM) digestion approach

- Transformation using digested and ligated Puc19 from the Mastermix digestion approach L(MM)

- Transformation using digested and ligated Puc19 from the No Mastermix digestion approach L(No MM)

- Positive control set up transformed E. coli bacteria with undigested and unligated Puc19 plasmid)

From the results we obtained after transformation as seen above, we had no growth for the following experimental setups:

- Transformation using direct digested Puc19 from the No Mastermix (No MM) digestion approach

- Negative control set up transformed E. coli bacteria with undigested and unligated Puc19 plasmid

1.6 CONCLUSION

This lab experiment we performed gave us the results that we envisioned. The results implied that the Mastermix digestion approach that we used did not ensure that all or some of the plasmids were digested, hence the growth from the direct transformation. We then concluded that using the No Mastermix approach for digestion for future lab experiments would be ideal.

2.0 SECOND LAB EXPERIMENT WITH SYNTHESIZED PROJECT PARTS

We conducted the second lab experiment with the rest of the synthesized parts for our project. We used the 1:10 concentrated plasmid, No Mastermix approach for digestion and made new competent cells for this last experiment with the final set of the synthesized parts.

We used the same reagents and micro volumes from the first lab experiment conducted with synthesized genetic parts for the project.

The results we obtained from the transformations are displayed below.

![](https://static.igem.wiki/teams/4186/wiki/fig-9.png)

Figure 9: Results obtained from Digestion and ligation of synthesized IDT and Twist parts (a) Gold inducer – Kill switch module (b) Arsenic inducer – Kill switch module (c) Ferric Inducer module

From the results displayed above, we observed a few colonies but still with low transformation efficiency. However, we took some of these colonies and grew them in the media overnight. We then used this overnight culture to perform a mini prep. After the mini-prep, we performed a Polymerase chain Reaction to amplify the inserted genes for each of the synthesized DNA in the Puc19 plasmid.

2.1 PCR EXPERIMENT

For the PCR, we had two tubes from two colonies of the Fe (I & II), two tubes from two colonies of Au and three tubes from three colonies of the As. We included a Puc19 plasmid DNA as control. Below are tables for PCR conditions and PCR reagent volumes we used.

![](https://static.igem.wiki/teams/4186/wiki/pcr-conditions.svg)

![](https://static.igem.wiki/teams/4186/wiki/pcr-reagents.svg)

2.2 GEL ELECTROPHORESIS

After the PCR, we run a gel with the amplified DNA parts. Below is a gel image of the gel run on the PCR amplified genetic parts.

![](https://static.igem.wiki/teams/4186/wiki/fig-10.jpg)

Figure 10: Gel electrophoresis results obtained for amplified DNA parts

2.3 CONCLUSION AND TROUBLESHOOTING

The anticipated band size for the genetic inserts used in this PCR gel are listed below.

![](https://static.igem.wiki/teams/4186/wiki/band-sizes.svg)

From the gel image above, it can be clearly observed that none of our genetic inserts were present (columns 2-8). However, the amplified portion of the puc19 in the PCR, which had approximately 184 base pairs, was present in each of the genetic inserts.
We suspected that one of the reasons the result came out the way it did might be because of the annealing temperature used for the PCR which was very close to the TM of the primers, impeding the amplification of the insert. Other reasons could be that the ligation of the parts might not have worked properly. We think that this is a possibility because the number of colonies was small. Since the plasmid cannot ligate itself we think that maybe few plasmids were not digested and lead to small transfection efficiency.

To trouble shoot if the PCR did not work correctly, we aimed to run another gel with the DNA parts directly from the mini prep. Unfortunately, we ran out of SYBR gold stain, used to visualize the DNA in the gel. We ordered some SYBR gold, but it did not arrive before the deadline to perform the experiment and hence could not troubleshoot this reasoning.

### FUTURE WORKS

📝 Once we have DNA staining dye, we would change the annealing temperature to increase the difference between the annealing temperature and TM .

📝 We would order new genetic parts for our project and troubleshoot digestion and ligation of the parts with the Puc19 plasmid.

### HYDROGEL ENCAPSULATION

Our solution proposed the deployment of live E. coli cells in the environment, so we ensured biosecurity and safety while maximizing the efficiency of our biosensor through hydrogel encapsulation. During the project's progress, the team moved from theoretical research to experimental lab work to test the theory of hydrogel1 encapsulation. Below is the protocol used and the results obtained. We could not add the hard shell of polyacrylamide hydrogel because our order did not arrive in time. Alginate forms hydrogel in di-cationic solutions (e.g., Ca2+, Ba2+) and has been used for various biomedical applications.

![](https://static.igem.wiki/teams/4186/wiki/fig-11.png)

Figure 11: Cell encapsulation in hydrogel capsules (a) schematic of the DEPCOS platform. (b) The process of core shell encapsulation of cells. Droplets of 2.5% alginate with engineered E. coli were crosslinked in a CaCl2 solution to form the soft core of the bead. Outsourced from (Tang, Tham, Liu, & Lu, 2020)

Manufacturing the Alginate Cores

A 10% alginate solution was made by dissolving medium viscous alginate in Distilled water, followed by autoclaving at 120 degrees to ensure sterility. A fresh bacterial culture with GFP and Red Mcherry was then mixed with the alginate solution in a one-to-one volume ratio to reach a final alginate concentration of 5 w.t%. This bacteria alginate premix was loaded into a syringe and dropwise added into a beaker of a CaCl2 solution (Fig. 11a) to form a beadlike droplet (Fig. 11b) with controllable diameters ranging from 2.5mm to 3mm. Immersing the droplet in the CaCl2 solution for a few minutes solidified the droplets formed.

![](https://static.igem.wiki/teams/4186/wiki/fig-12.png)

Figure 12: (left) The process of core shell encapsulation of cells. 2.5% alginate solution (right) Droplets of engineered E. coli crosslinked in a CaCl2 solution to form the soft core of the hydrogel

![](https://static.igem.wiki/teams/4186/wiki/fig-13.png)

Figure 13: Hydrogel containing E coli cells with (a) alginate containing cells with GFP (b) alginate containing cells with Red mcherry (c) Alginate

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