iGEM 2023 — Team the-kings-school-au

{% 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

Term 1 — March to April

## Session 1 - 1st March

This was our first session together as a group. We listened to a presentation from Chris Yoo and Pranav, who gave us a brief insight into how iGEM worked and their journey last in 2022. After this, we were introduced to the idea of synthetic biology and how it is a new tool allowing us to manipulate the genes of organisms to make them do useful things to solve a problem. We had a quick look at some past iGEM projects. This session was just understanding what we were undertaking.

## Session 2 - 8th March

This was another theory-heavy session, however, we did get to start using pipettes, which was a fun experience for everyone. We learned about the registry of parts in iGEM and bio-bricks, extending our knowledge of synthetic biology. We also look at how ordering DNA in the sequence you specify has become ever cheaper with time at only 2 cents per base pair in places. We then learned about the different sizes of puppets, how to load and unload the tips, and the proper pipetting technique. Thi set up an activity where we had to pipet a certain volume of coloured water onto a scale and see how accurate we were.

## Session 3 - 15th March

We had an introduction to microbiology by learning about different bacteria and how we can select certain ones of an agar plate using antibiotics. We watched a video on how to create the agar mixture and how to pour an agar plate. We then learned the different methods of inoculating an agar plate which is streaking and spreading. We attempted to draw a picture on an agar plate using different coloured bacteria.

## Session 4 - 22nd March

This was our first plasmid extraction. We started the lesson learning about plasmids as rings of DNA that are replicated in the cytoplasm of cells. These plasmids can then be extracted separately from the nucleic DNA and cellular matter. This is important because we can replicate DNA by inserting it into a cell and then extracting the copies later.

![](https://static.igem.wiki/teams/4735/wiki/dnaextraction.png)

Term 1 Holiday Block — April

## Session 1 - 19th April

Robert from Hydgene Renewables gave us a university-level summary of how cells are used in synthetic biology, and we experimented with SnapGene, a gene modelling software application. We learnt about the different sections of a plasmid, such as promotors and termination points. DNA replication was also a large portion of the lesson since this related directly to why we used cells in synthetic biology — to make copies of the DNA/RNA we want. Finally, we also learnt about protein-coding and wrote our names in codons which represent proteins and letters of our name.

Term 2 — May to June

## Session 1 — 3rd May

During this session, we learnt about the way to extract the highest quality Dna from the plasmids of the bacteria cells. Additionally, we took notes on how this trials is properly conducted, with single and double cut extractions having different steps involved.

![](https://static.igem.wiki/teams/4735/wiki/restrictionenzyme.png)
![](https://static.igem.wiki/teams/4735/wiki/singledoublecut.png)

## Session 2 — 10th May

During this session we learnt how to cast an Agarose Gel, in order to conduct electrophoresis and separate the different sizes of DNA base pairs (b.p). The gel was noted to be kept at a temperature of 55°C during the cast to keep it as a liquid, and is to be stored at 4°C. The liquid gel is poured into the mould and allowed to set. It is then submerged in the electrophoresis machine and allowed to run for 20 mins.

![](https://static.igem.wiki/teams/4735/wiki/agarosegel.png)

## Session 3 — 17th May

This session had a greater focus on the entrepreneurial side of our project, beginning with a short video by Simon Sinek. The TED talk detailed the principles of pitching ideas to buyers, whether it be corporations or masses of customers, and emphasised the importance of “selling your idea” as opposed to selling just a product. Sinek made use of the Golden Circle model, dictating how the “Why” aspect of the product is the most influential factor in decision making for the buyer, as opposed to facts and statistics (the “What”) which don’t register with the decision making part of the brain. Another concept was the Law of Diffusion of Innovation, which dictated the portions of a population that will seek to buy your product at different stages. Sinek used the model to show how it is important to sell your product to early adopters, so that the majority of the population will be more inclined to buying the product, resulting in market success. He linked this back to his idea by stating that it is the idea, the “Why”, which appeals to the early adopters.

## Session 4 — 24th May

We began brainstorming project ideas. This was done in groups of 4, first we brainstormed potential ideas for our project, many of the concepts related to nature. Afterwards, we narrowed it down to: soil health, different types of pollution including water, air and land, restoring mines, clothes waste, biomass (continuing last year’s project) and more. In our groups, we prioritised as a team what we felt were the most important issues to address in our current world in science, rather than what would be the best project. This was done by placing the ideas in a numbered list from most important to least important, discovering what our fellow teammates were passionate about. The general consensus of all groups ended settling on the top problems of soil health, mining, and plastic waste.

## Session 5 — 31st May

To finish this term, we conducted another plasmid extraction. We started the lesson by revising our knowledge of plasmids as rings of DNA that are replicated in the cytoplasm of cells. These plasmids can then be extracted separately from the nucleic DNA and cellular matter. This is important because we can replicate DNA by inserting it into a cell and then extracting the copies later. Each of the students involved were competing against each other to achieve the cleanest and highest quality extraction.

Term 2 Holiday Block — July

## Session 1 — 10th July

We had an extremely productive day, fleshing out our project, assigning roles, and making progress towards iGEM. Louise and Robert gave us their ideas on how we could fix ammonia directly from hydrogen, bypassing the entire nitrogen fixing part. This is far more efficient. We were asked to bring in soil samples from around where we live and we analyses them qualitatively and also by measuring the pH.

The soil was mixed with water in a ratio of 1:9 and shaken for 30 mins. 50 ml of the soil mixture was inoculated into a liquid media solution and 10 microlitres was inoculated into a semisolid solution. If ammonia is produced, these solutions will changes colour from green to blue. This indicates the presence of diazotrophs. The inoculation was repeated for every soil sample and also repeated for the entire set using different carbon sources in the solutions. E.g Glucose, NxFree malate or mannitol. This allows us to see which ones the diazotrophs like the most.

Finally, we decided on rough working groups so people could specialise in what they liked doing the most. Documentation going forward will be a group effort, with each person logging their work throughout the day in a Google Form. Tom Hanley will create a summary at the end of each session and maintain the logbook using the entries from the Google Form.

![](https://static.igem.wiki/teams/4735/wiki/twelvesoilsamples.png)
![](https://static.igem.wiki/teams/4735/wiki/bunsenburner.png)
![](https://static.igem.wiki/teams/4735/wiki/shakingmachine.png)

## Session 2 — 11th July

### Wet Lab

We made 4 types of agar plates,( glucose , sucrose, malate, mannitol ) carbon sources. A total of 25 plates are made, with 2 sets of 12. One set labelled with H (high), One set labelled with L (low). The plates are labelled as the following, ( Date , type of carbon source, plate number, plate type)

1:1000 ratio for high concentration - 499.5 μL of media and 0.5 μL of sample solution
1:10^-6 ratio for low concentration - 499.5 μL of media, 0.5 μL of high concentration sample corresponding (HC 1 to LC 1, etc)
100 μL of finished plasmid was loaded onto agar, then spread. To spread, glass rods were sterilised using ethanol and lighting, and after cooling we smeared the samples.

### Business and Marketing

During today's session, we completed many different goals. During the first two hours, we researched companies in the market of promoting soil health and their existing products, and created a name for the team. In the next two hours, we set up the Instagram page for the project, and continued researching the impacts that our project could have. During the last two hours, we edited the grant for the NSW Government, Interview Questions for experts interviews, and brainstormed collaborators and sponsors.

### Media and Programming

Tully created a beautiful logo of a tree with its trunk being the DNA and extra sprouts coming off it as the roots. He created all different versions of the logo, including black and white ones with and without text. Tom replicated Tully's drawings in Adobe Illustrator and is now animating them in Adobe After Effects. This will be used as the landing page on the website. Ahmed helped Tully with the design of the logo and is also brainstorming ideas for other animations on the website such as the loading screen. Tully is also storyboarding Tom's animations and website ideas.

### Dry Lab

We researched soil quality indicators, formatted the document, and began a literature review. Found that diazotrophs release more ammonia in root environments due to nitrogen fixation. Replacing glutamine synthetase with a repressible promoter could help. However, issues with uAT mutants outcompeting desired diazotrophs were observed. Multiple uAT copies showed some success in hydroponic and non-fertilized soil setups. This info may be useful for improving diazotrophs if initial tests are unsatisfactory.

![](https://static.igem.wiki/teams/4735/wiki/foiledconicalflasks.png)
![](https://static.igem.wiki/teams/4735/wiki/hughkimwriting.png)
![](https://static.igem.wiki/teams/4735/wiki/renqiagarplates.png)

## Session 3 — 12th July

### Business and Marketing

There were many different business and marketing efforts that were completed today. In the first two hours, we revised and finished the government grant and published the first post on the Instagram account. During the next two hours, we organised interviews with experts and external companies and organised the "Meet the Team" posts for Instagram. In the final 2 hours, we organised photos with the King's Marketing Department and started organising the promotional video for the iGEM competition.

### Dry Lab

Today we continued on from yesterday's goal and finished the similar project section of the literature review alongside continuing the "cause of soil health defects" section. Sir gave us a new article to consider in terms of design, and this will add much to the analysis in our literature review.

We investigated the mechanism of action for Nitrogen fixation in Diazotrophs, focusing on the well-known MoFe mechanism. We identified the chemical responsible for nitrogen reduction during fixation to NH3. Additionally, we pinpointed the key structural proteins (MoFe and Fe), understood their roles and cellular positions, and explored the nif genes responsible for coding the MoFe (nifD and nifK for alpha and beta subunits) and Fe (nifH) proteins, along with various accessory and regulatory nif genes. Our research delved into the functions of accessory proteins aiding structural protein assembly and the regulation of Nitrogen fixation by regulatory proteins (nifL, nifA). We also acquired skills in gene sequence retrieval using specialized software, considering the target bacteria for expression. Concurrently, Anthony made progress in researching environmental factors impacting soil health, including alternative soil health indicators and their connections to diazotrophs and bacteria.

### Design and Programming

Photographic documentation of the liquid and semi-solid solutions was conducted. The samples are really starting to show tinges of blue now. The website landing page was tweaked, changing the colour of the DNA from red and blue to green and light green. Tom then worked on creating the Wiki. He ran into troubles when it came to SSH signing and cloning the GitLab repository. He is now using GitHub in the meantime in combination with Visual Studio Code. We also spent a bit of time choosing Wiki styles and looking at the due dates for each of the iGEM deliverables.

![](https://static.igem.wiki/teams/4735/wiki/greenvials.png)

## Session 4 — 13th July

### Business and Marketing

Today was a very productive day. In the first two hours, we finished brainstorming various different ways to communicate our research and posted our first "Meet the Team" on Instagram. In the next two hours, we contacted the media management team at Incitec Pivot fertilisers, and finished planning the promotional video. In the final two hours, we interviewed David Payne about soil health and fertilisers and distributed promotional posters around the school.

### Media and Programming

Productive day! Ahmed and Tully made SOILutions posters (A3 & A4), placed around the science center. Tom improved Agar plate photo technique using a 45-degree angle lightbox. Uploaded pics to Google Drive with new naming. Tom also worked on Wiki landing page. Still waiting for iGEM team acceptance. Created GitHub repo and used Visual Studio Code. Ahmed designing a comic book for human practices. Tully collaborated on social media posts. Tom took team photos with Marketing. Ideal location: second floor, science center, wooden panels.

## Session 5 — 14th July

### Wet Lab

Sara and Marcus worked on the DNA and oxygen sensor prototype design. We looked through papers to find the DNA sequence we needed for our experiments. Then we copied and pasted the DNA sequence in a document for organisation. When all the parts were in and ready to order we went to the Biolabs’s website and pasted the parts on their order page to make it for our wet lab team to use. However, at first, the sequence was too complex to order for the company so we ordered the parts separately to reduce the complexity at their attainable level.

### Media and Programming

Tom enhanced the website landing page with a sliding sidebar for smaller screens. Marketing scripted a 2-minute promo video, approved by lab teams. Tom set up recording gear, found Lab 5 as the best location, and used cinematic mode on iPhone 13 Pro. A TFP friend acted as a teleprompter. Tully, Daniel, Erica, and Skanda presented well. Tom edited audio and made a prototype video. Planning to use Adobe After Effects for sleek transitions and animations. Tom and Daniel discussed additional visuals for the video. At 3 pm, Tom's mum was interviewed by marketing, providing soil analysis insights. The TFP holiday session was highly productive, setting the stage for term 3. Deadline: the first day of term 3 holiday session for iGEM deliverables.

![](https://static.igem.wiki/teams/4735/wiki/deniedorder.png)
![](https://static.igem.wiki/teams/4735/wiki/reqparts1.png)
![](https://static.igem.wiki/teams/4735/wiki/reqparts2.png)
![](https://static.igem.wiki/teams/4735/wiki/goldengate1.png)
![](https://static.igem.wiki/teams/4735/wiki/goldengate2.png)

## Session 6 - 16th July

Tom enhanced the website's responsiveness with a sliding sidebar. The marketing team crafted a 2-minute video script, validated by wet and dry lab teams, and we received clearance to begin recording. Tom brought recording equipment, utilized cinematic mode on an iPhone 13 Pro, and found Lab 5 as the ideal location. A former TFP member acted as a teleprompter. Tully, Daniel, Erica, and Scanda presented seamlessly. Tom edited audio and created a promising video prototype, planning to use Adobe After Effects for added sleekness. At 3 pm, Tom's mother, experienced in soil and plant sample analysis, provided insights for soil analysis but limited input on the synthetic biology aspect. We wrap up our session well-prepared for Term 3, aiming to complete iGEM deliverables by the start of the Term 3 holiday session.

## Session 7 - 19th July

We focused on rewriting the promo video script and planned a re-recording session for week three due to issues with cinematic mode and presentation. The recording will take place in the more visually appealing P2 Lab. Tully created another Meet The Team Instagram post for the Design and Programming team, and we captured photos of absent team members. Tom made adjustments to the website and photographed liquid, semi-solid samples, and Agar plates using a lightbox. Ahmed and Tully began storyboarding an animated landing page for the website, designed to guide viewers through our gene design, with Tom exploring its feasibility.

## Out of Session Event - Interview with Dr Charles Day

Today we interviewed Dr Charles Day, who is the CEO of Jupiter Ionics. There were a lot of general talks; however, we did get a couple of gems that changed the way we think about our project. They are using a non-biological solution for the production of ammonia, particularly nitrogen in the soil. Recently, demand for their products has gone up with the fertiliser shortage, but they are not yet ready to produce in a commercial sense. We then posted an Instagram post about our interview.

![](https://static.igem.wiki/teams/4735/wiki/charlesday.jpg)

Term 3 — July to September

## Session 1 - 19th July

### Wet Lab

Ian Helped with the project design – continued labelling parts of the DNA. We took out the agar plates to take photos. Create agar plates for samples we ordered to prepare for experiment next week.

### Marketing and Programming

We worked on rewriting the script for the promo video. We also want to record it since the fake blur of cinematic mode is not quite nice, and the presentation by some people was not to their liking. In week three, Tom will bring his recording equipment again to redo the promo video. We will be recording it in the P2 Lab, which has far more interesting scenery. Tully worked on creating another Meet The Team Instagram Post for the Design and programming team. We also took photos of other team members who were absent during the holiday session. Tom worked a bit more on the website, reshuffling some files. He also took photos of the liquid and semi-solid samples. Using a lightbox, Tom also took photos of the Agar plates. Ahmed and Tully started storyboarding the website's landing page, which will be an animation to scroll position type effect that takes the viewer through our gene design. Tom will look further into the feasibility of this. Today we also finalised our additional questions for our interview with Dr Charles Day tomorrow, and we coordinated another "Meet the Team" Instagram post. Additionally, we coordinated our fun facts, facts.

## Session 2 — 26th July

### Wet & Dry Lab

We practiced the polymerse chain reaction technique to prepare for the next session where we would undertake the PCR procedures for our DNA.

### Marketing and Programming

Robert gave us a talk on the parts that we've created. Three are three parts, with two having three subparts. There is a Tspacer followed by a promotor. There is a ribosome binding site after the promotor means we make lots of protein. You can change the promotors and rbs to modify the amount of protein/RNA. The terminator tells RNS polymerase to stop transcription. The protein that is being made is called "Green Fluorescent Protein" which will cause the cells to glow green when a UV light is shined on them. There is also an FNR gene which is a DNA-binding protein, and it can sense how much oxygen there is. This allows more RNA to be produced when the oxygen is at the right threshold and restricts it when the oxygen is not at a suitable level. We are going to put some of these bacteria in an anaerobic chamber and some exposed to air. We hope to see that the bacteria in the anaerobic chamber will glow brighter since more of the protein will be produced. This would indicate the oxygen sensor is working. We are also trying three different promotors to find which one produced the least protein under high oxygen conditions. At first, we will complete this qualitatively, and then we can move on to quantitative measurement. Today we had five parts made. These are from the start to the TSpacer, The next part was the green fluorescent protein. We are going to use golden gate assembly to put the parts together, making the longer strand we want. We use a restriction enzyme to cut the DNA in a specific shape which means another piece can attach itself to it. BSC I is the restriction enzyme we are using. We will then take these genes and put them in bacteria and hope the bacteria start fluorescing.

## Session 3 — 2nd August

### Wet Lab

Our wet lab team was working on loading the 14 samples into the gel for it to run. And Sara was pipetting the samples for it to mix with the purple dye (which Ella worked on to pipette). Hugh was loading the samples to the lanes one by one. Eventually, gel ran and we looked at it using UV light box (Macus helped this) but there was no fluorescent on the gel and we also didn't put the ladder in.

Conducted interview with Jason Simmons, started creating a survey for consensus. Started the 'meet the team' component for the Wiki. ALSO adding photos of soil sample (Number 6).

![](https://static.igem.wiki/teams/4735/wiki/soilsample.png)

## Session 4 — 10th August

### Wet Lab

Golden Gate Assembly: We inserted our parts into the framework of the original DNA using the golden gate assembly, using 3 different NifA parts ordered.

### Marketing and Programming

The marketing team took official and professional photographs for all members of the team, to be used on the team wiki. Additionally, also refilmed the promotional video with an updated script.

## Session 5 - 16th August

Furthered progress on meet the team for dry and wet lab, continued adding onto wiki sentences for group members, worked on human practices questions for wiki, provided details for animations for our promotional video.

## Session 6 - 13th September

We finished and posted our survey, finished the wiki descriptions for the team roster, and furthered our information contained in the human practices section

Term 3 Holidays — October

## Session 1 - 4th October

### Programming

Resident web engineers - Adrian Zhuang and Tom Hanley - began moving the team’s research and progress onto the team wiki online. The programming team decided to begin work on the team’s notebook page, and Adrian began migrating the team’s past session notes online, using an expandable accordion tab to display session notes grouped into monthly intervals.

We also compiled a list of all the wiki pages requiring creation and assigned each member to work on their respective content within a shared Word document. The web engineers will subsequently transfer this content to the website. Tom primarily focused on developing the website's homepage, utilizing SVGs to design waving dividers and enhance the appearance of images and text. The homepage mirrors the flow of the promotional video and features a scroll-based animation at the outset, contributing to a cohesive and appealing presentation.

![](https://static.igem.wiki/teams/4735/wiki/adriantom.jpg)

### Wet and Dry Lab

Test 6 constructs of their fluorescent behaviour in 4 different conditions (anaerobic, aerobic, 2% Oxygen level and 0.5% oxygen level)-Renqi and Ella filled 29 tubes with base CM and separated them into 7 groups. The 7th group which is the negative control includes 5 tubes 6 of them contain 4 tubes, each tube contains 10 microliter samples (1A,3A,2B,4B, 2C and 1C). After that, we sealed 1 tube from every group (7 tubes) to make them in anaerobic condition and placed 1 tube from every group (7 tubes) into each desiccator. Then, we send two desiccators to the lab to create 2% oxygen level and 0.5% oxygen level environment for the constructs by filling the right amount of air.

![](https://static.igem.wiki/teams/4735/wiki/groupphoto1.jpg)

{% 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

### Content

1. [Design](#design)
2. [Build](#build)
3. [Test](#test)
4. [Learn](#learn)
5. [Citations](#citations)

## Design

### Objective

To effectively implement diazotrophic nitrogen fixation in E.coli using the Nitrogenase pathway and ensure its optimal operation under varying oxygen conditions.

### The Mechanism

* Diazotrophs: Specialised bacteria capable of converting atmospheric nitrogen (N2) to bioavailable ammonia (NH3).
* Nitrogenase Pathway: Employed predominantly by diazotrophs, this pathway revolves around two core structural proteins - Reductase (sourced from nifH) and Nitrogenase (from nifD & nifK). To facilitate the pathway, additional regulatory genes and proteins are essential, with nifA playing a regulatory role in overseeing nitrogenase protein synthesis.

### Challenges and Solutions

Oxygen Sensitivity: When interacting with oxygen, nitrogenase can generate reactive oxygen species (ROS) which can be harmful to the cell, thus, ensuring reasonable efficiency mandates careful regulation in response to oxygen concentrations, with levels above 2% O2 considered detrimental. Our solution thus lies in harnessing an inherent oxygen-sensitive system in e.coli, fumarate and nitrate reductase (FNR), to upregulate transcription in optimal conditions while deactivating in high oxygen, reducing transcription thus ensuring cell safety.

### Test Structure

Our designs tested 3 promoters, narGp, narKp and dmsAp each modified to possess an extra half binding site [TTGAT] (1.5 total binding sites each) with the intention of increasing FNR sensitivity to maximise yield in low O\_2 conditions.

With the final intention to have FNR upregulate nifA, our test structure utilised green fluorescent protein (GFP) as a placeholder for nifA while varying the promoter interacting with FNR to optimise yield. In addition to differentiating via the intensity of fluorescence (indicating rate of transcription), different oxygen levels were also tested ensuring transcription is adequately oxygen-sensitive and is sufficiently reduced with >2% O\_2 concentration.

### Part Design

Our designs tested 3 promoters, narGp, narKp and dmsAp each modified to possess an extra half binding site [TTGAT] (1.5 total binding sites) with the intention of increasing FNR sensitivity to maximise yield in low O\_2 conditions.

Our oxygen prototype consisted of the following 3 parts (parts 2 and 3 were put together in the registry):

* Part 1 constant (BBa\_K4735012): lacIQ promoter which provides baseline rate of transcription for rbs-fnr followed by a TrrnBT1 terminator. This provides a constant supply of FNR protein
* Part 2 variable (BBa\_K4735013, BBa\_K4735014, BBa\_K4735015 (all include part 3)): A T-Spacer and T7 binding site (for future positive control testing), followed by the promoter to which FNR will bind, each containing 1.5 binding sites, varies between a narGp, narKp and dmsAp promoter.
* Part 3 constant: Ribosome binding site (different to the rbs in part 1) rbs2 followed by superfolder fgfp and then a terminator TrrnBT1.

### Primers for PCR amplification + Golden Gate Assembly

For assembly, we utilised the NEBridge golden gate assembly tool to design primers for PCR, yielding:

* Prototype A = Part 1(ac) + Part 2a(a) + Part 3(ac)
* Prototype B = Part 1(b) + Part 2b(b) + Part 3(b)
* Prototype C = Part 1(ac) + Part 2c(c) + Part 3(ac)

Where: Prototype n = Part 1(primer) + Part 2n(primer) + Part 3(primer)

## Build

### Delivery of parts and amplification via PCR

After performing a quality check confirming the quality of delivered parts with gel electrophoresis, PCR protocol was followed to amplify parts 1, 2, and 3 for prototypes A, B, and C with primers indicated above, after which another gel electrophoresis was performed confirming parts were correctly amplified.

### Golden Gate Assembly

We mix the our parts together in the molar ratio described in the kit, however, the provided vector was not used, and instead a 1:1:1:1 ratio of Part 1, Part 2, Part 3, and pSB1C3 (amplified BioBrick Vector with BsaI sites) was used.

We followed golden gate assembly protocol to assemble our plasmid as per the ratio above and incorporated this into our transformant NEB 5-alpha competent E.coli cells.

As a final quality check, we screened transformants and extracted plasmid DNA, which was tested via gel electrophoresis and 3rd party sequence verification.

## Test

### Qualitative Observations

Utilising handheld UV testing on agar plates, the viability of the GFP in prototype cells was confirmed by observing fluorescence.

### Qualitative Experiments

Our e.coli was incubated into liquid culture set to desired atmospheric oxygen levels of 0% (anaerobic), 0.5%, 2%, and aerobic conditions (~18%). Once cells were given time to incubate bacteria growth was measured using a microplate reader, then the fluorescence intensity of GFP at each condition was recorded. Analysis was then performed adjusting for relative intensity yielding a graph, normalised to OD600, portraying the relative fluorescence of each promoter compared to a control with no GFP (A, B, and C is NarG, NarK, and DmsA respectively).

![](https://static.igem.wiki/teams/4735/wiki/gfpintensity.jpg)

Fig 1: GFP intensity normalised to OD600

## Learn

Our experiment shows that our test cycle succeeded as FNR sensitivity of oxygen was shown to be successful as an oxygen sensor with promoters B (NarK) and C (DmsA) whereas it showed no O2 sensitivity in promoter A (NarG). As shown on the graph, high fluorescence intensity was observed when our prototypes were incubated in oxygen conditions below 2%. For the purposes of our project as a whole, we’ve determined that promoter B of NarK is most suitable for the continuation of our project as it exhibited more stable, consistent fluorescence across different oxygen intervals with a more compact spread of results than promoter C (DmsA) between the biological replicates.

![](https://static.igem.wiki/teams/4735/wiki/promotorresponsetooxygen.jpg)

Fig 2: Alternate expression of GFP fluorescence to oxygen concentration.

As seen in the graph above, there is an increase in GFP production in both successful promoters from 0% to 0.5%. Also, both successful prototypes became inactive at ~18% oxygen approaching atmospheric conditions as expected. Hence, both systems successfully function as an oxygen sensor to the requirements of our experiment.

### Future Endeavours

Following the success of FNR oxygen sensitivity, our next step is to substitute the sfGFP indicator with NifA, which regulates nitrogenase production. We believe that this will maximise the efficiency of nitrogenase production, by optimising its production to only occur when nitrogenase can function, under the ideal oxygen concentration levels. Through maximising the production of nitrogenase, we should be able to efficiently produce ammonia within the soil, effectively revitalising the ammonia content of the soil in a self-sustaining manner.

After incorporating the NifA regulator, future efforts could focus on developing a sensor to other soil factors, such as carbon-source availability, and concentration of minerals such as Iron and Molybdenum that are required in the process of nitrogen fixation. By successfully optimising our E.coli to be able to sense a multitude of environmental conditions, the efficiency of nitrogen fixation can be greatly enhanced.

## Citations

Ryu et al., (2020). Control of nitrogen fixation in bacteria. Nature Microbiology.

Kiley, P. J., & Beinert, H. (1998). FNR's role in oxygen sensing. FEMS Microbiology Reviews.

Martinez-Argudo, I., et al. (2004). The multifaceted NifL-NifA System.

{% 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 %}

# Experiments

### Content

1. [Soil Sample Investigation](#soilSampleInvestigation)
2. [Plasmid DNA Cutting](#plasmidDNA)
3. [Electrophoresis](#electrophrosis)
4. [Plasmid Extraction](#plasmidExtraciton)
5. [Golden Gate Assembly](#GoldenGate)
6. [GFP Fluorescence Intensitys](#GFP)

## Soil Sample Investigation

### Materials

* 1. 12 different soil samples
* 2. Semi-solid agar
* 3. Agar
* 4. Distilled Water
* 5. Glucose, sucrose, malate, and mannitol broth
* 6. Bromothymol Blue Indicator

### Protocol

1. 1. Each of the 12 soil sample were diluted with 100 ml of distilled water.
2. 2. Diluted samples were placed on an orbital shaker at 80 RPM for 15 minutes
3. 3. Samples were inoculated into semi solid and solid agar containing bromothymol blue indicator.
4. 4. 500 µl of each sample was also added to a 25 ml mix of bromothymol blue indicator and a broth of each carbon source (glucose, sucrose, malate, mannitol).
5. 5. All samples were incubated at 25°C, and were periodically removed to be inspected and analyzed.

![](https://static.igem.wiki/teams/4735/wiki/shaker.jpg)

Fig 1.1: Preparation of diluted soil samples to be introduced to media

## Plasmid DNA Cutting

### Materials

* DNA
* Buffer 2.1
* 2uL of EcoRI
* water
* 1uL of Pst 1

### Protocol

1. Water is combined with the chosen DNA amount.
2. 10x the amount of DNA is added in buffer fluid.
3. The restriction DNA is added to the solution.

### IF Single Cut

1. 1. 80ng of the chosen DNA is added to the solution.
2. 2. 1uL of EcoRI is added to the solution.
3. 3. 10x the amount of DNA is added in buffercutsmart.
4. 4. Water is added to the solution.

### IF Double Cut

1. 1. 80 ng of the chosen DNA, times by x uL.
2. 2. 1uL of EcoRI is added to the solution.
3. 3. 1uL of Pst I is added to the solution.
4. 4. 10uL of buffer 2.1 fluid is added to the solution.
5. 5. Water is added to the solution to dilute.

## Electrophoresis

### Materials

* Electrophoresis tray
* Comb
* Agarose
* TAE buffer
* sticky tape
* Electrophoresis chamber
* DNA samples
* pipette
* Ultraviolet light

### Protocol

1. 1. Seal the edges of an empty agarose gel tray with masking tape to prevent leakage during gel preparation.
2. 2. Place a comb with evenly spaced teeth into the tray, determining the location and size of sample wells.
3. 3. Carefully pour liquid agarose (kept at around 55 degrees Celsius) into the tray up to the new top of the comb's teeth.
4. 4. Allow the agarose to solidify for approximately 20 minutes.
5. 5. Gently remove the spacing comb from the tray, leaving wells for loading samples.
6. 6. If not used immediately, store the gel at 4 degrees Celsius.
7. 7. Place the solidified agarose gel into the electrophoresis chamber.
8. 8. Pour electrophoresis buffer solution into the chamber, ensuring it covers the entire gel surface and fills the chamber to an adequate level.
9. 9. Prepare the DNA samples you wish to test. These samples may come from various sources or experiments.
10. 10. Using a syringe or a dedicated pipette, suction up each DNA sample, ensuring a clean, sterile tip for each sample.
11. 11. Carefully add the DNA samples into the wells created by the comb during gel preparation. Insert the tip of the syringe or pipette into each well to keep the DNA inside the well without floating in the buffer liquid.
12. 12. Attach the chamber's lid securely to maintain the electric field within the chamber and prevent sample evaporation.
13. 13.Connect the electrophoresis chamber to the power supply.
14. 14. Turn on the power supply unit.
15. 15. pply an electric field that causes the DNA molecules to migrate through the agarose gel. The rate of migration depends on the size and charge of the DNA fragments.
16. 16. Run the electrophoresis at a specified voltage (e.g., 100V) for a designated duration (commonly around 1 hour, but it can vary based on gel thickness and DNA fragment size).
17. 17. After the electrophoresis run, turn off the power supply and disconnect the chamber.
18. 18. Carefully remove the gel from the chamber.
19. 19. Place the gel on a UV transilluminator or gel documentation system.
20. 20. Observe the separated DNA bands, which have migrated based on their size, under ultraviolet (UV) light. Document the results as necessary.

## Plasmid Extraction

### Materials

* Centrifuge
* 2ml E. coli culture
* 250 µl Suspension Buffer
* 250 µl Lysis Buffer
* 350 µl Binding Buffer
* High Pure filter tube
* 500 µl of Wash Buffer I
* 700 µl of Wash Buffer II
* 100 µl of Elution Buffer

### Protocol

1. 1. Centrifuge 2ml or E. coli culture at 6,000 RPM for 30 seconds.
2. 2. Discard supernatant (the leftover liquid that is not the pellet).
3. 3. Resuspend the pellet in 250 µl of Suspension Buffer.
4. 4. Add 250 µl of Lysis Buffer
5. 5. Mix gently. Incubate for 5 mins at room temperature.
6. 6. Add 350 µl of chilled Binding Buffer.
7. 7. Mix gently. Incubate for 5 mins on ice. Centrifuge for 10 mins at max speed.
8. 8. Discard the pellet by pouring the remaining liquid into a High Pure filter tube and discarding the original tube with the pellet at the bottom.
9. 9. Centrifuge at max speed for 30-60 seconds.
10. 10. Discard flowthrough
11. 11. Add 500 µl of Wash Buffer I.
12. 12. Centrifuge at 13,000 RPM for 1 minute.
13. 13. Discard the flowthrough
14. 14. dd 700 µl of Wash Buffer II.
15. 15. Centrifuge at 13,000 RPM for 1 minute.
16. 16. Discard flowthrough
17. 17. Centrifuge at 13,000 RPM for a further 1 minute.
18. 18. Discard flowthrough.
19. 19. Add 100 µl of Elution Buffer.
20. 20. Centrifuge at 13,000 RPM for 1 minute.
21. 21. The liquid in the bottom is what we want to keep.

## Golden Gate Assembly

### Prototype A

BBa\_K4735004 BBa\_K4735005 BBa\_K4735006 BBa\_K4735009 BBa\_K4735010 BBa\_K4735003

narG promoter driving superfolder GFP expression.

The part should promote the expression of GFP under low oxygen conditions.

Testing indicates this does not respond very well to oxygen. It shows relatively high GFP fluorescence under all oxygen conditions.

Prototype A, will consist of :
Part 1 - lacIQ-rbs-fnr-TrrnBT1
Part 2 - T-Spacer-T7-3FNRbindingNarG (part a)

### Prototype B

BBa\_K4735004 BBa\_K4735005 BBa\_K4735007 BBa\_K4735009 BBa\_K4735010 BBa\_K4735003

FNR under control of lacIQ promoter for testing of narK promoter driving superfolder GFP expression.

The part should promote the expression of GFP under low oxygen conditions.

Testing indicates this does respond very well to oxygen. It shows no GFP fluorescence anaerobically or aerobically but gives GFP fluorescence at 0.5% oxygen and 2% oxygen.

Part 1 - lacIQ-rbs-fnr-TrrnBT1
Part 2 - T-Spacer-T7-3FNRbindingNarK (part b)
Part 3 - RBS2-sfgfp + TrrnBT1

### Prototype C

Prototype C will consist of :

BBa\_K4735004 BBa\_K4735005 BBa\_K4735008 BBa\_K4735009 BBa\_K4735010 BBa\_K4735003

FNR under control of lacIQ promoter for testing of dmsA promoter driving superfolder GFP expression.

The part should promote the expression of GFP under low oxygen conditions.

Testing indicates this does respond to oxygen. It shows no GFP fluorescence aerobically AND gives GFP fluorescence anaerobically at 0.5% oxygen and 2% oxygen in nitrogen.

Part 1 - lacIQ-rbs-fnr-TrrnBT1
Part 2 - T-Spacer-T7-3FNRbindingDmsA (part c)
Part 3 - RBS2-sfgfp + TrrnBT1

## GFP Fluorescence Intensity under Varying Oxygen Concentrations

### Materials

* LB media
* 29 tubes
* Labels for tubes
* Negative control sample
* Anaerobic chamber
* Desiccator
* Loosely screwed caps for tubes
* High vacuum grease
* Syringe
* Balloon
* Cylinder filled with water
* Nitrogen gas
* Rubber stopper
* Gas chromatography machine
* Shaker

### Protocol

In this experiment, we picked 6 samples (1A, 3A, 2B, 4B, 1C, 2C) and 1 negative(L) control to grow in 4 different conditions (aerobic, 0.5% oxygen, 2% oxygen, anaerobic ). Each 50 ml falcon tube is labeled in the following way:
condition(Ae, 0.5%, or 2%, An), sample name(1A, 3A, 2B, 4B, 1C, 2C, or L), LB(the media)

1. 1. Add 10 µl samples from each of the six constructs to the corresponding tubes. (eg. add 1A sample to all tubes with label 1A on it)
2. 2. Add 10 µl each sample from negative control to each of the tubes.
3. 3. Create an anaerobic chamber by vacuum sealing one chamber, and an aerobic chamber full of standard air.
4. 4. Place the 7 tubes labeled with 0.5% with loosely screwed caps into the chamber of desiccator and place 7 tubes labeled 2% with loosely screwed caps into another chamber of desiccator.
5. 5. Seal the desiccators by applying high vacuum grease on the edge of the container and the lid and valve.
6. 6. Purge the desiccators by flowing nitrogen gas through for 1 minute.
7. 7. Calculate the volume of the gas (air) to fill into the desiccator to make a 0.5% oxygen condition (using c1v1=c2v2).
8. 8. Use a syringe to measure 50 ml of air to inflate the balloon, mark the estimated volume on the surface of a big cylinder filled with water above the waterline and test the volume of the balloon by immersing the balloon in the water to observe which it reaches the mark.
9. 9. Open the valve before filling the nitrogen gas to the desiccator by connecting the tube from the gas tank to the tap of the desiccator and slide the cover slightly open to let the air out
10. 10. Squeeze the tube tightly to ensure no gas leakage and open the rubber stopper quickly and inject the air into the chamber by connecting the syringe to the balloon and closing it immediately after injection.
11. 11. To fasten the equilibrating process, use the empty syringe into the tap and purge at least 10 times
12. 12. Once equilibrated, insert the GC needle into the rubber stopper and draw 5 µl gas from the chamber and put it into the gas chromatography which will automatically calculate the actual concentration of oxygen % to ensure there is the correct amount of oxygen in the desiccator.
13. 13. Repeat steps 6-12 again for the 2% desiccator(for step 8 and 9, insert 4 times as much air as the amount inserted into the 0.5% desiccator).
14. 14. If the percentage of oxygen in the desiccator is accurate, seal the desiccators and the anaerobic chamber, incubate them and the aerobic tubes at 37 degrees with 90 RPM speed shaking overnight.
15. 15. Create two biological replicates and a technical triplicate sample for each sample under the varying oxygen concentrations.
16. 16. Using a gel reader, measure GFP intensity per OD600 to model the response of each sample to oxygen.

![](https://static.igem.wiki/teams/4735/wiki/purging.jpg)

Fig 2.1: Purging of desiccators to create 0.5, 2% oxygen chambers

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{% extends "layout.html" %}
{% 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

### Content

1. [Soil Survey Results](#soilsurvey)
2. [GFP Flourescence Results](#GFPFlourescence)
   * [Qualitative Testing](#qualitativetesting)
   * [Methods](#method)
   * [Quntitative Results](#quantitativeResults)
   * [Graphing Results](#graphingResults)
   * [Further Prototype B Data](#furtherPrototype)
   * [Future Plans](#futureplans)

## Soil Survey Results

An investigation into potential diazotroph presence was conducted to gain a better insight into the current impacts of nitrogen fixation in the soils of varying local regions. By surveying and testing 12 different soil samples from differing geographical locations, biological activity, human impact, and composition, and the effect of different bacteria in these soils, we were able to conclude that diazotrophs not only were a viable solution that could be implemented to local Australian soil; furthermore it was highly likely that species of diazotrophs were already present, and fixating nitrogen already in some of our soil.

This experiment observed the changing pH of a range of media that were inoculated with bacteria from the wide variety of soil samples. These media consisted of semi-solid agar, agar plates, and liquid broths containing bromothymol blue indicator to visualise the different survivability and prevalence of such bacteria in glucose, sucrose, malate, and mannitol sources as media.

The media turning from green to blue indicated that it became more basic, potentially due to the production of ammonia. Hence, a sample that turned blue hinted at the likelihood of nitrogen fixation occurring. Contrastingly, a change in colour to yellow shows the samples were turning more acidic, signifying the fermentation and growth of bacteria, as this would result in a release of carbon dioxide gas which would in turn increase the acidity of the sample.

![](https://static.igem.wiki/teams/4735/wiki/identificaitonofsoilsampletable.jpg)

Fig 1.1: Identification of soil samples

![](https://static.igem.wiki/teams/4735/wiki/semisolidagartable.jpg)

Fig 1.2: Semisolid agar colour change after 1 week, relatively scaled (0- no change, 1- slight change, 2- intense change)

![](https://static.igem.wiki/teams/4735/wiki/semisolidsamples.jpg)

Fig 1.3: Photo of semisolid agar colour change after 1 week.

![](https://static.igem.wiki/teams/4735/wiki/sugartypestable.jpg)

Fig 1.4: Relative colour change of liquid media after 1 week (+ for blue change, scale of 1 to 3 for intensity, - for yellow change, 1 to 3 for intensity)

![](https://static.igem.wiki/teams/4735/wiki/mannitolcolorcchange.jpg)

Fig 1.5: Photo of Mannitol 7-12 colour changes after 1 week

## GFP Fluorescence Results

### Qualitative Testing

Handheld UV testing, a UV light was used to check for GFP fluorescence on the Agar plate which we incubated our assembled Prototypes. Fluorescence would indicate that our prototype worked and allowed us to advance in more detailed testing.

![](https://static.igem.wiki/teams/4735/wiki/uvlight.jpg)

Fig 2.1: Qualitatively testing GFP flourescence using a UV handheld torch.

Then E coli is incubated into liquid culture at set desired atmospheric oxygen levels of anaerobic, 0.5% oxygen, 2% oxygen and aerobic conditions. Once cells are given time to incubate, bacteria growth is measured using a microplate reader. Then the fluorescence intensity of GFP at each condition will be recorded. Finally, analysis will be done by adjusting the relative intensity per bacteria growth and a graph will be generated to allow for further learning.

The test result will first allow us to determine whether our prototype is “oxygen sensitive” and secondly to determine which prototype variant ( A, B, C ) is best, with the most focused response and the largest range.

### Methods

#### Preparing liquid construct

For each oxygen level, pour 20ml of set liquid construct (eg prototype A) into 50 tubes. Then repeat for the other two prototypes ( B and C). Then pour 20 ml of liquid nutrient without construct as control. In total, there should be 7 tubes of culture for each oxygen level and 28 in total.

#### Preparing set oxygen levels

Anaerobic conditions
To achieve anaerobic conditions, test tubes are placed within a sealed container with a hand warmer acting as a catalyst to use up the oxygen present in the container. Then the container is placed on a shaker set at 37\*C and 90 rpm.

0.5% oxygen conditions
To achieve 0.5% oxygen conditions, a vacuum desiccator was first filled with Nitrogen to purge all oxygen out of the container. Then 5 ml of oxygen is introduced through the tap. Then a gas chromatography test is run to verify the percentage of oxygen. Then the desiccator is seal-clamped and placed on a shaker set at 37\*C and 90 rpm.

2% oxygen conditions
To achieve 2% oxygen conditions, a vacuum desiccator was first filled with Nitrogen to purge all oxygen out of the container. Then 29 ml of oxygen is introduced through the tap. Then a gas chromatography test is run to verify the percentage of oxygen. Then the desiccator is seal-clamped and placed on a shaker set at 37\*C and 90 rpm.

Aerobic conditions (16% oxygen)
Test tubes are placed on a test tube holder on a shaker set at 37\*C and 90 rpm.

#### Loading Construct in microplate for imaging

Two biological replicates were grown overnight, and 150 µL of each culture was put into a 96-well plate in triplicate, and the plate’s OD600 was measured. The GFP fluorescence (485 nm Excitation, 520 nm Emission) was also measured in a BMG-Pherastar plate reader at a gain setting of 400. By imagining the samples, the bacteria growth of each sample could be quantified which would be useful for understanding the GFP fluorescence relative to the amount of bacteria present.

![](https://static.igem.wiki/teams/4735/wiki/bacteriabrowthtable.jpg)

Fig 3.1: Bacteria growth data, imaged at 595 wavelength
Row A-B: Anaerobic conditions. Row C-D: 0.5% oxygen growth conditions. Row E-F: 2% oxygen growth conditions. Row G-H: Aerobic (18% oxygen) growth conditions. A1-6 NarG tests, A7-12 NarK tests, B1-6 DmsA tests, B6-9 Empty plasmid. Pattern repeats for remaining conditions. Aerobic extra control H10-12.

#### Measuring GFP fluorescence

GFP fluorescence data is recorded using a plate reader, 2 Biological replicants were taken for each Prototype and technical replicates for each value.

### Quntitative Results

\*\*The values of the blank were subtracted from the raw data, and normalised for OD600 measurement.

![](https://static.igem.wiki/teams/4735/wiki/rawdataflourescencemeasurments.jpg)

Fig 3.2: Raw Data (485/520) GFP Fluorescence measurement of same plate. Row A-B: Anaerobic conditions. Row C-D: 0.5% oxygen growth conditions. Row E-F: 2% oxygen growth conditions. Row G-H: Aerobic (18% oxygen) growth conditions. A1-6 NarG tests, A7-12 NarK tests, B1-6 DmsA tests, B6-9 Empty plasmid. Pattern repeats for remaining conditions. Aerobic extra control H10-12.

![](https://static.igem.wiki/teams/4735/wiki/normaliseddata.jpg)

Fig 3.3: Normalised data to OD600 and blank subtracted. Row A-B: Anaerobic conditions. Row C-D: 0.5% oxygen growth conditions. Row E-F: 2% oxygen growth conditions. Row G-H: Aerobic (18% oxygen) growth conditions. A1-6 NarG tests, A7-12 NarK tests, B1-6 DmsA tests, B6-9 Empty plasmid. Pattern repeats for remaining conditions. Aerobic extra control H10-12.

![](https://static.igem.wiki/teams/4735/wiki/summaryofdata.jpg)

Fig 3.4: Summary of fluorescence data per sample category.

![](https://static.igem.wiki/teams/4735/wiki/oxygenvsgfpflourescence.jpg)

Fig 3.5: Oxygen % vs GFP fluorescence.

### Graphing Results

Data collected above is generated into a graph to allow better understanding. The GFP fluorescence is adjusted according to bacteria growth per optical density at 600.The GFP intensity scale is normalised to 0 according to our Control, due to background fluorescence.

\*\* Promotor A was taken out due to its abnormal reaction that we cannot explain at this point
\*\* We will do research and re-experiment with promoter A in the future

![](https://static.igem.wiki/teams/4735/wiki/gfpintensitynormalised.jpg)

Fig 3.6: GFP intensity normalised to OD600

Thus our experiment shows that our test cycle succeeded as FNR sensitivity of oxygen is proved to be successful as an oxygen sensor with promoters B (NarK) and C (DmsA) whereas it showed no O2 sensitivity in promoter A (NarG). As shown on the graph, high fluorescence intensity was observed when our prototypes were incubated in oxygen conditions below 2%. For the purposes of our project as a whole, we’ve determined that promoter B of NarK is most suitable for the continuation of our project as it exhibited more stable, consistent fluorescence across different oxygen intervals with a more compact spread of results than promoter C (DmsA) between the biological replicates.

![](https://static.igem.wiki/teams/4735/wiki/alternateexpressionofgfpflourcescence.jpg)

Fig 3.7: Alternate expression of GFP fluorescence to oxygen concentration

As seen in the graph above, there is an increase in GFP production in both successful promoters from 0% to 0.5%. Also, both successful prototypes became inactive at ~18% oxygen approaching atmospheric conditions as expected. Hence, both systems successfully function as an oxygen sensor to the requirements of our experiment.

### Further Prototype B Data

![](https://static.igem.wiki/teams/4735/wiki/promotorbbreponsetooyxgen.jpg)

Fig 4.1: NarK Promotor response to verying oxygen concentrations

### Future Plans

Following the success of FNR oxygen sensitivity, our next step is to substitute the sfGFP indicator with NifA, which regulates nitrogenase production. We believe that this will maximise the efficiency of nitrogenase production, by optimising its production to only occur when nitrogenase can function, under the ideal oxygen concentration levels. Through maximising the production of nitrogenase, we should be able to efficiently produce ammonia within the soil, effectively revitalising the ammonia content of the soil in a self-sustaining manner.

After incorporating the NifA regulator, future efforts could focus on developing a sensor to other soil factors, such as carbon-source availability, and concentration of minerals such as Iron and Molybdenum that are required in the process of nitrogen fixation. By successfully optimising our E.coli to be able to sense a multitude of environmental conditions, the efficiency of nitrogen fixation can be greatly enhanced.

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