Contribution

An overview of what the 2024 UAlberta iGEM team has to offer for future teams

Project Description Engineering Success Future Directions
Utilization of RBS1_12000 (BBa_K5427000) Tutorial Videos Music Videos

Utilization of RBS1_12000 (BBa_K5427000)

Our team focused on the characterization of RBS1_12000, and conducted a combination of experiments designed to measure its effectiveness in a variety of constructs and conditions. These experiments evaluated bacterial growth, fluorescent protein production, and the impact of salt concentration on metabolic efficiency, providing a deep dive into the analysis of RBS1's utility in synthetic biology applications.

In experiments testing the pTac_RBS1_sfGFP construct, RBS 1_12000 consistently showed the best overall growth in most E. coli strains (DH5α, BL21, K12, and Rosetta Gami) when compared to other ribosome binding sites. When optical density (OD600) was measured every two hours for a total of 10 hours, the data indicated that RBS 1_12000 promoted robust bacterial growth in all tested strains when compared to empty strain. Importantly, RBS 1_12000 showed no impact on bacterial proliferation at both 30oC and 37oC, suggesting a strong ability to support efficient protein synthesis under diverse growth conditions (Figures 1-4).

Fig 1. | Growth curve of pTac_RBS1/2/3/4_sfGFP_pJUMP24 vector in 4 different E. coli bacterial strains; A) DH5ɑ, B) BL21, C) K12, and D) Rosetta-gami. These strains were transformed with our constructs, grown at 30oC and growth quantified using optical density (OD) at 600. OD measurements were taken every 2 hours for a total of 10 hours for each culture while growing in liquid LB.
Fig 1. | Growth curve of pTac_RBS1/2/3/4_sfGFP_pJUMP24 vector in 4 different E. coli bacterial strains; A) DH5ɑ, B) BL21, C) K12, and D) Rosetta-gami. These strains were transformed with our constructs, grown at 30oC and growth quantified using optical density (OD) at 600. OD measurements were taken every 2 hours for a total of 10 hours for each culture while growing in liquid LB.
Fig 2. | Growth curve of pTac_RBS1_sfGFP_pJUMP24 vector in 4 different E. coli bacterial strains; A) DH5alpha, B) BL21, C) K12, and D) Rosetta-gami. All 4 strains were transformed with our construct and measured for growth and 30oC using optical density of 600 nm to measure the growth of each strain. OD measurements were taken every 2 hours for a total of 10 hours for each culture while growing in liquid LB.
Fig 2. | Growth curve of pTac_RBS1_sfGFP_pJUMP24 vector in 4 different E. coli bacterial strains; A) DH5alpha, B) BL21, C) K12, and D) Rosetta-gami. All 4 strains were transformed with our construct and measured for growth and 30oC using optical density of 600 nm to measure the growth of each strain. OD measurements were taken every 2 hours for a total of 10 hours for each culture while growing in liquid LB.
Fig 3. | Growth curve of pTac_RBS1/2/3/4_sfGFP_pJUMP24 vector in 4 different E. coli bacterial strains; A) DH5ɑ, B) BL21, C) K12, and D) Rosetta-gami. These strains were transformed with our constructs, grown at 37oC and growth quantified using optical density (OD) at 600. OD measurements were taken every 2 hours for a total of 10 hours for each culture while growing in liquid LB.
Fig 3. | Growth curve of pTac_RBS1/2/3/4_sfGFP_pJUMP24 vector in 4 different E. coli bacterial strains; A) DH5ɑ, B) BL21, C) K12, and D) Rosetta-gami. These strains were transformed with our constructs, grown at 37oC and growth quantified using optical density (OD) at 600. OD measurements were taken every 2 hours for a total of 10 hours for each culture while growing in liquid LB.
Fig 4. | Growth curve of pTac_RBS1_sfGFP_pJUMP24 vector in 4 different E. coli bacterial strains; A) DH5alpha, B) BL21, C) K12, and D) Rosetta-gami. This strain was transformed with our construct and measured for growth and 37oC using optical density of 600 nm to measure the growth of each strain. OD measurements were taken every 2 hours for a total of 10 hours for each culture while growing in liquid LB.
Fig 4. | Growth curve of pTac_RBS1_sfGFP_pJUMP24 vector in 4 different E. coli bacterial strains; A) DH5alpha, B) BL21, C) K12, and D) Rosetta-gami. This strain was transformed with our construct and measured for growth and 37oC using optical density of 600 nm to measure the growth of each strain. OD measurements were taken every 2 hours for a total of 10 hours for each culture while growing in liquid LB.

Further supporting its efficiency, fluorescence data revealed that RBS 1_12000 produced a significantly stronger fluorescence signal than other tested RBSs, confirming its superior role in driving the expression of superfolder green fluorescent protein (sfGFP). The fluorescence signal of RBS 1_12000 was measured at 10.33 units when probation expression was induced for 3 hours using 3 mM IPTG, in stark contrast to the signals of RBS 2, 3, and 4, which were -0.66, -4.66, and -2.66, respectively. When protein expression was induced overnight with 1 mM IPTG, we saw an increase in fluorescence for RBS 1 at 105 units whereas no significant increase in fluorescence was detected for RBS 2/3/4. This demonstrates that RBS 1_12000 resulted in an increase in sfGFP production by 1665.15% when compared to RBS 2, underscoring its high efficiency in translation initiation (Figure 5 and Figure 6).

Fig 5. | Fluorescence (with blank subtracted) data for sfGFP isolated from DH5a containing the pTac_RBS 1/2/3/4_sfGFP_pJUMP 24 plasmid after 3 hour induction with 3 mM IPTG.
Fig 5. | Fluorescence (with blank subtracted) data for sfGFP isolated from DH5a containing the pTac_RBS 1/2/3/4_sfGFP_pJUMP 24 plasmid after 3 hour induction with 3 mM IPTG.
Fig 6. | Fluorescence (with blank subtracted) data for sfGFP isolated from DH5a containing the pTac_RBS 1/2/3/4_sfGFP_pJUMP 24 plasmid after overnight induction with 1 mM IPTG.
Fig 6. | Fluorescence (with blank subtracted) data for sfGFP isolated from DH5a containing the pTac_RBS 1/2/3/4_sfGFP_pJUMP 24 plasmid after overnight induction with 1 mM IPTG.

The J23119_RBS1_pJUMP 28 construct was also tested under different salt conditions using Lennox LB (low salt) and regular LB to investigate the impact of sodium concentration on bacterial growth. Furthermore, when metabolic burden of J23119_RBS1_pJUMP 28 was tested through growth curves in different strains of E. coli (DH5α, BL21, and Rosetta Gami) over 10 hours, RBS 1_12000 still supported adequate growth in both conditions (Figure 8), with some variation. In regular LB, the growth was slightly better, with BL21 showing a 16.6% increase in growth at the 10-hour mark compared to Lennox LB. DH5α also exhibited a 29% increase in growth under regular salt conditions (Figure 8). These results suggest that while salt concentrations can influence growth, RBS 1_12000 is well-suited to promote protein expression in both standard and low-salt environments without significant inhibition of bacterial growth.

Fig 7. | Growth curve for J23119_RBS1_pJUMP28 in E. coli strains DH5alpha, Rosetta-gami, and BL21. Cultures were grown for 10 hours at 37oC in regular LB media, and measured for optical density (OD) every 2 hours at 600nm. Each culture was repeated 3 times and its averages were plotted on a growth curve for analysis.
Fig 7. | Growth curve for J23119_RBS1_pJUMP28 in E. coli strains DH5alpha, Rosetta-gami, and BL21. Cultures were grown for 10 hours at 37oC in regular LB media, and measured for optical density (OD) every 2 hours at 600nm. Each culture was repeated 3 times and its averages were plotted on a growth curve for analysis.
Fig 8. | Growth curve for J23119_RBS1_pJUMP28 in E. coli strains DH5alpha grown in regular LB or Lennox LB (low salt condition) at 37oC. Each culture was grown for 6 hours, and optical density (OD) was measured every 1.5 hours at 600nm.
Fig 8. | Growth curve for J23119_RBS1_pJUMP28 in E. coli strains DH5alpha grown in regular LB or Lennox LB (low salt condition) at 37oC. Each culture was grown for 6 hours, and optical density (OD) was measured every 1.5 hours at 600nm.

Our contribution is recorded on the iGEM parts registry as Part: BBa_K5427000 (RBS 1_12000). This entry contains the summarized contributory data for this part and can be here iGEM Parts Registry
.

Tutorial Videos

UAlberta: Aseptic Techniques (2024) [English]

In this aseptic techniques tutorial video, you'll be guided through the fundamental practices required to maintain sterility in the lab and prevent contamination. The video explains key procedures such as sterilizing equipment, working within a sterile field, proper handwashing. You'll also learn best practices for using tools like Bunsen burners and laminar flow hoods to ensure a contaminant-free environment. Perfect for those working in microbiology, cell culture, or clinical labs, this tutorial provides the essential knowledge to maintain aseptic conditions in any experiment.

UAlberta: Micropipetting Tutorial (2024) [English]

In this micropipetting tutorial video, you'll learn the essential techniques for accurate and precise liquid handling in a laboratory setting. The video covers step-by-step instructions on how to properly use a micropipette, including setting the volume, loading and dispensing liquids, and avoiding common errors like air bubbles and contamination. Ideal for beginners or those needing a refresher, this tutorial ensures you develop the skills needed for efficient and reliable pipetting in any scientific experiment or research environment.

Music Videos

The UAlberta iGEM Extended Play bridges the field of science with the voice of music through creating a sonic world about our project and the power of microbiology, interpreting the sounds of the lab and our project into music. An incredibly large thank you to the highly talented Ian Maxwell Spotify
for taking our thoughts into a new artistic space.

UAlberta: Micro Groove (2024) [English]

"Microgroove" is an infectious, feel-good track that pulses with rhythm and energy. It captures a smooth, flowing vibe that pulls listeners in, layering vibrant beats with a seamless blend of retro and modern sounds. The track has an easygoing yet dynamic pace, making it perfect for both a chill evening or an energetic gathering. Its rhythm weaves in and out with precision, creating an immersive, hypnotic experience that keeps listeners hooked from start to finish.

UAlberta: Synthetic Silk (2024) [English]

"Synthetic Silk" is a sleek, atmospheric track that glides effortlessly, enveloping the listener in a smooth, dream-like soundscape. It’s both calming and hypnotic, with an underlying pulse that gives it a subtle momentum. The song feels like floating through a futuristic world, with delicate layers that shimmer and evolve throughout. Its gentle pace creates a sense of tranquility, while its intricate rhythms add a touch of sophistication, making it both relaxing and captivating, like silk slipping through your fingers.

UAlberta: Lab Work (2024) [English]

"Lab Work" crackles with energy, a sharp and crispy track that snaps into action with a tight, fast-paced rhythm. Its crisp beats and sudden shifts give it an edge, like sparks of inspiration firing in a busy lab. Each section moves with precision, creating a sense of urgency and discovery. The track stays lean and focused, with an almost mechanical feel, reflecting the calculated intensity of experimentation. It’s a perfect backdrop for moments of high concentration, where every detail counts, and the next step could lead to a breakthrough.

UAlberta: Change (2024) [English]

"Change" is an electrifying, high-speed rush that never slows down. From the first second, it launches into a whirlwind of rapid beats and unpredictable shifts, mirroring the chaotic energy of transformation. The track feels alive, constantly evolving, with no time to pause—pushing forward at a relentless pace. Its intensity creates an exhilarating sense of motion, like being caught in the middle of a storm where everything is shifting in real-time. "Change" is all about momentum, leaving listeners breathless as it accelerates toward an unpredictable finish.

Bonus: UAlberta memepage

For when the hours were long and the days were not long enough, we kept our spirits up.