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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
We practiced the polymerse chain reaction technique to prepare for the next session where we would undertake the PCR procedures for our DNA.
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.
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).
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.
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.
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.
We finished and posted our survey, finished the wiki descriptions for the team roster, and furthered our information contained in the human practices section
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.
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.
