Engineering Success

Engineering Success

Overview

Deciding on a Topic

Our process began with a broad brainstorming session involving the entire team, focusing on problems we could address through genetic engineering. We explored various ideas, such as plant methane metabolism (enhanced methane removal via plant genetic editing), airborne toxin lung filtration (using genetically modified microorganisms to detect and neutralize harmful airborne toxins before they enter the respiratory system), plastic decomposition, lead poisoning therapeutics, and fungi soil resilience increase (using bioengineered fungi to increase soil resilience).

During our brainstorming session, we heavily researched these ideas and discussed each one based on its potential usefulness, impact, and uniqueness. We wanted to choose a project that would make a significant difference while also standing out in its approach.

Our results on each topic were as follows:

Plastic Decomposition

• Has been done extensively in the past.
• Our approach would likely involve editing algae or E. coli to decompose plastic.
• Many plastics already have decomposition methods, so we’d need to choose a unique target. Initially, we considered PVC, but it proved too toxic to work with.

Removing Lead from Lungs

• A unique idea with significant potential impact.
• However, experimentation would be challenging due to the complexity of working with human biology.
• It would be difficult to create a solution more effective and cheaper than EDTA.

Airborne Toxin Filtration (Nitrous Oxide)

• This was an incredibly useful idea, given that nitrous oxide is a potent greenhouse gas and a significant contributor to air pollution.
• We planned to edit E. coli to remove nitrous oxide from the air, which would be valuable in areas with high industrial emissions.
• However, we found that an iGEM team had already attempted something similar, limiting the novelty of our approach.

Methane Removal

• Methane is another potent greenhouse gas that significantly impacts global warming, making its removal from the atmosphere highly beneficial.
• We aimed to edit plants to absorb methane from the air, starting with E. coli as a proof of concept.
• The use of the methane monooxygenase enzyme was especially promising because, at the time, no one had yet edited E. coli with this enzyme.

We ultimately narrowed down our focus to removing greenhouse gasses from the environment, but we found it challenging to choose between Airborne Toxin Filtration (targeting nitrous oxide) and Methane Removal. Both ideas had significant potential for reducing harmful emissions and addressing climate change. For about a month, we invested a considerable amount of time researching both approaches.  

Change of Plans

With methane removal, our initial plan was to  genetically modify an ornamental tobacco plant (commonly used in genetic edits) to absorb methane. This plant could be placed near oil factories and farms to remove methane from the air. Since plants naturally take in large amounts of air, this approach seemed more efficient than using regular methanotrophic bacteria. We planned to start by editing E. coli and later move on to the plant.

However, in December, a study came out performing the same experiment. The scientists also attempted inserting methane monooxygenase into e coli! It was exciting to see scientists with the same idea as us perform the experiment, but it meant we were back to the drawing board.

We revisited our earlier idea involving nitrous oxide. We considered the possibility of genetically editing a plant to remove both methane and nitrous oxide, as they are often found in the same environments. However, we soon realized that executing both genetic edits would be too time-consuming, especially since it was our first time as an iGEM team. Ultimately, because our idea for airborne toxin filtration with nitrous oxide was to genetically modify plants to turn nitrous oxide into nitrogen via nitrous oxide reductase, which was not a novel idea in the scientific community, we shifted our focus to methane completely.

As we researched more on methane, we identified landfills as the third-largest source of methane emissions. Methanotrophs, or methane-eating bacteria, would not be effective in this environment due to their limited ability to survive and thrive in landfill conditions. This led us to consider Bacillus subtilis, a bacterium commonly found in soil and far more abundant than typical methanotrophs.

Design 1

Following the steps a group from the University of Korea did, we replicated the work done in https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11116448/ to build the protein sequence for the mini-sMMO. From there, we used IDT’s codon optimizer to optimize for B. subtilis, resulting in our final DNA sequences for the mini-sMMO alpha and beta subunits.  

This document contains a summary of the mini-sMMO article, the protein sequence for the mini-sMMO, and the DNA sequence for the mini-sMMO.

We designed a construct using that sequence with Leah, a graduate student from the University of Wisconsin's, help.

Our final linear DNA is composed of 4 main fragments: a pDR110 segment, the mini-sMMO alpha subunit, the mini-sMMO beta subunit, and another pDR110 segment. (see first diagram). The front and the back of our linear DNA product is amyE back and amyE front, which are the homologous parts of our DNA that allow it to replace the amylase gene of B. subtilis’s chromosomal DNA.

Build 1

We PCRed fragment one and fragment four from PDR110, and ordered fragment two and fragment three from IDT. There were challenges in that process, fragment two failed IDT’s quality checks, and so they had to send it to us in two parts. To address this we ran overlap extension PCR to combine the two parts IDT gave us, and then tried to combine all our parts using Gibson assembly.

 

(Left to right): ladder, fragment 1 (2.6k), blank, ladder, fragment 4 (2.1k)

Using a NEB 1 kb+ ladder.

Test 1

We decided to split our testing into four groups. Two sets, each with a different glycerol stock were created. Within each set we transformed PCRd and non PCRd versions of the Gibson Assembly. Unfortunately we got zero transformants. To test further, ran a gel on our PCRd gibson, which led us to a realization: Our PCR results were 3000 bp shorter than expected.

 

[left to right] PCRd gibson, NEB 1 kb+ ladder

Learn 1

We started considering what went wrong, and then we realized the 4.6kb PCR product lined up with the size of just fragment one and fragment four, without our insert. So we tried to consider what could have produced a result of that size.

We eventually realized that the primers we were using would amplify any DNA starting with amyE back and ending with amyE front, including our plasmid (PDR110). Our plasmid likely entered our Gibson from the unpurified fragment one and fragment four.

Design 2

We started to rethink our lab protocol, with help from Leah. The major changes we made were:

• Start with PCR purified DNA.
• Not PCRing the gibson to avoid amplifying portions of PDR110.
• Circular DNA doesn’t transform well, so plasmid leftovers should be fine.
• Use simpler competent cell prep

Build 2

We returned to the lab again, re-running PCR on all of our fragments (there are four official ones, but fragment 2 was shipped to us in two parts by IDT giving us a total of five DNA sequences to amplify. We ran three reactions of each, ran a gel on ten ul of each, and pooled the remainder for PCR purification. We then ran a five way Gibson assembly, and transformed the results into our newly prepared competent cells.

 

[from right to left] ladder, frag 1, frag 2a, frag 2b, frag3, frag 4, frag 1, frag 2a, frag 2b, frag 4, blank, frag 1, frag 2a, frag 2b, frag 4.

Zoomed in version of above only including [from right to left] ladder, frag 1, frag 2a, frag 2b, frag3, frag 4

Test 2

This time we had transformants! However when we let the plates grow longer we also ended up with colonies on our negative control. To confirm that the colonies were actually successful transformants, we plated them on new plates with starch and added iodine. If transformation was successful the bacteria would not be able to consume tha starch (amylose) due to a knocked out amylase gene.

Iodine reacts with starch to turn purple, and otherwise remains yellow, so successful transformants should have purple covers, whereas unsuccessful ones should have yellow/orange hallows.

 

Learn 2

We learned the hard way that spectinomycin, our antibiotic, actually degrades significantly over the course of a week at 37 C, resulting in contamination. Our starch test results are not conclusive, so we would like to run further tests to confirm or deny the production of sMMO. We also hope to confirm that our Gibson reaction actually worked by sending it in for sequencing.

In the end, by following the engineering cycle we were able to go from a vision in our heads of B. subtilis absorbing methane to live transformants of genetically engineered B. subtilis in the lab. While we do still have additional lab work to do, we believe this along with our work on hardware demonstrates engineering success.