Contributions

The iGEM Munich 2024 team has undertaken ambitious projects with a focus on innovation and community impact. The contributions go beyond the specific project goals, with the aim to benefit future teams and researchers working in synthetic biology.

Characterization of Mutant Promoters in DH5a E. coli

Summary

In the wet lab, our team conducted rigorous promoter strength measurements to better understand and quantify gene expression levels. Specifically, we focused on the family of constitutive promoter parts BBa_J23100 through BBa_J23119, originally isolated from a combinatorial library by the iGEM Berkeley 2006 team, which has been widely used for synthetic biology applications (Florea et al., 2016). In 2006, the Berkeley Arkin Lab integrated these promoters into the pSB1A3_J61002 plasmid . Subsequently, in 2008, Jason Kelly from the same group designed two additional promoters, BBa_J23150 and BBa_J23151, by introducing point mutations in BBa_J23107 and BBa_J23114, respectively. However, these novel promoters have not been characterized yet.

In our study, we investigated the impact of these mutations on promoter strength in E. coli. While many studies utilize electrocompetent cells, such as TG1, we opted to explore the performance of these promoters in chemically competent DH5α cells, which are known for their cost-effectiveness despite having lower transformation efficiency compared to electrocompetent cells. This approach could allow more accessible and knowledgeable usage of these promoters in less expensive workflows.

Methodology

Our experiments built on comparison with the study by the Tongji China iGEM team, which found BBa_J23114 to be the most efficient promoter in DH5α. We used the KLD (kinase-ligase-dephosphorylation) technique to ligate a PCR-amplified fluorophore encoding plasmid (pSB1A3_J23106-mTurquoise-B10015 CDSmut) without its original promoter but with overhangs that allowed the incorporation of BBa_J23150 and BBa_J23151 promoters upstream of the fluorophore coding region.

Results

J23150p and J23150:
J23150pBBa_J23150 contains a point mutation in previously BBa_J23107 and the composite tested in has an additional nonsynonymous substitution in the CDS of mTurquoise(L9F) (mTurq), resulting increased promoter activity to 255% compared to BBa_J23104, surpassing this previously strongest tested promoter of the promoter family. Reversion of the mTurq point mutation to test J23150 without the influencing fluorophore mutation, resulted in an activity of 160% compared to BBa_J23104 and unmutated J23107 (50 %). This suggests that this point mutation might have a critical role in boosting promoter efficiency.
J23151:
The J23151 promoter, derived from BBa_J23114, showed an increase in activity to 343% compared to BBa_J23104, higher than the original promoter (BBa_J23114) of 14% (XY) in comparison to BBa_J23104. This highlights the profound impact of the mutation in promoter strength enhancement.

Conclusions

The findings characterize the existing promoters, designed by iGEM06_Berkeley group in 2006, to investigate the influcence of single point mutiations, alowwing future teams to take highly informaed choices about their apromoters.

The improvements observed in promoters BBa_J23150 and BBa_J23151 in comparison to the well-characterized BBa_J23104 reference demonstrates influence on profound biological effects via minimal synthetic mutagenesis of regulatory genetic elements of the . This allows for the development of more efficient expression systems and synthetic biology constructs generally.

By characterizing two existing, uncharacterized promoters, we provide valuable data for other synthetic biologists aiming to precisely control gene expression in their systems and optimize genetic circuits that require precise regulation of protein production or overall efficiency. Our work also serves as a foundation for future teams aiming to design reliably tunable gene circuits.

Methods

Fluorescence Measurement

Measurements were conducted in E. coli DH5α strain, in biological triplicates and technical duplicates. Successfully transformed E. coli were defined by Whole Plasmid Sequencing, for point mutation reversal by Sanger Sequencing.

Colonies were picked from the transformation plate and grown overnight for 12-16 h in LB + Carbenicillin (50–100 µg/mL), reaching ~ OD600 0.8 - 1.2 the next day. Cells were spun down ~ 2.5 min per mL inoculation medium at 4°C and 4000 rpm and resuspended in the same volume PBS.

Fluorescence was measured in a well plate with a plate reader.

96 well plate: 150 µL
mTurq: ex.: 434 nm; em.: 474 nm
initial 20 s at 300 rpm plate shaking, 10 s pause - start

The blank OD600 was subtracted from the OD600 of samples, and the emission at 474 nm was normalized to the individual OD600 values by dividing each fluorescence value by the sample OD600. The mean was calculated across all biological and technical samples, for each sample respectively.

Promoter – Fluorophore Reporter Cloning

Step by step and explaination of the KLD reaction

PCR Amplification with Overhangs:

The process typically starts with a polymerase chain reaction (PCR) to amplify the desired DNA fragments. The primers are designed with overhangs, which contain sequences corresponding to the ends of the DNA fragments to be joined and 3´ and 5´ ends respectively, that can be seemlesly ligated via KLD reaction, in sequence resembling the rquired promoter, in a fast way for small insertions.

In the case of our experiment, we amplified a plasmid minus the promoter but added overhangs that resemble the split promoter parts (e.g., BBa_J23150 or BBa_J23151), allowing these parts to ligate into the plasmid later.

Mechanistic example of the primer design for promoter exchange via KLD.

Kinase (Phosphorylation by T4 PNK):

The T4 polynucleotide kinase (PNK) adds 5’ phosphate groups to the ends of the DNA fragments. This phosphorylation is necessary because DNA ligase requires a 5’-phosphate group to join DNA fragments.

Normally, PCR products don’t have 5’ phosphates, so this step prepares the fragments for ligation by adding the missing phosphate groups.

Ligase (Joining DNA Fragments):

T4 DNA ligase then catalyzes the formation of phosphodiester bonds between the 3’ hydroxyl group of one DNA fragment and the 5’ phosphate of another. This process joins the DNA fragments, completing the circularization of the plasmid or the creation of a new recombinant DNA construct.

In our case, the ligase connects the overhangs corresponding to the promoter regions (e.g., BBa_J23150, BBa_J23151) to the plasmid, forming a complete, circular plasmid that includes the new promoter.

DpnI Digestion (Removing Template DNA):

DpnI is a restriction enzyme that specifically cuts methylated and hemimethylated DNA, which is characteristic of plasmid DNA isolated from E. coli strains. Since the original plasmid template used in PCR is methylated, DpnI digestion removes the template DNA.

This ensures that any remaining parental (template) plasmid is destroyed, leaving only the newly ligated (mutated or modified) DNA to be transformed into bacteria.

Transformation:

After the KLD reaction is complete, the newly ligated plasmid can be introduced into competent bacterial cells (such as DH5α or TG1 strains) via transformation. Once inside the bacteria, the plasmid replicates, and the transformed cells express the new recombinant construct.

Original Data /& Standard Piological Parts

Final Plasmid with insertion of J23150 upstream of mTurq.

For transparency, here you can find the original data of our obtained results:

Raw Data - Contributions

New (composite) parts resulting from our contribution efforts are available from BBa_K2722003, [BBa_K5102004] (https://parts.igem.org/Part:BBa_K5102004) and [BBa_K5102005] (https://parts.igem.org/cgi/partsdb/part_info.cgi?part_id=93780) to BBa_K2722008.

3D Print Your Own Central Dogma Puzzle Game

As synthetic biology rapidly evolves, there’s a growing need for more educational tools and resources to support different educational models. It’s no wonder that most iGEM teams, including iGEM Munich (check out more on our Education page), engage in educational outreach, especially with high school students, introducing key concepts like DNA, RNA, protein expression, and the central dogma of molecular biology. We find that the mechanisms of the molecular processes replication, transcription, and translation are often hard to visualize due to the dynamic nature of the processes. That is why, we designed a 3D-printed puzzle focused on the central dogma, which can be used by all future iGEM teams in their educational activities. The puzzle is designed to be highly customizable and adaptable to different size and age groups and can be implemented in workshops covering a wide range of molecular biology topics such as: mutations, protein expression, nucleic acids, properties of the genetic code, PCR and replication etc.

Here is a quick demonstration of the basic functionalities: https://video.igem.org/w/rxNbpuwiytUPoCWH5uhjCq

We developed, prototyped and tested the puzzle in several schools and educational activities and implemented the feedback that we got. We are happy to share the 3D models for future iGEM teams to implement in their educational workshops. You can download the 3d models ready for printing here, as well as the background files here:
Click here to download printable backgrounds ↓

We also created a detailed guide on how to print, assemble and use the puzzle game. The handbook features instructions for one standard cooperative game for a group of 20-25 students and seven additional variations of the game for bigger and smaller groups and varying difficulty levels with instruction for every one of them.

Here is the guide! https://static.igem.wiki/teams/5102/contribution/puzzle-files/handbook-3d-puzzle-munich2.pdf

We cannot embed the models on our wiki, but we are more than happy to provide them, so contact us at info@igem-munich.com ! Here are some pictures of the models:

Guide on 3D-Modeling Without Previous CAD Experience

When designing the puzzle, we stumbled on the problem that none of the team members had any experience with 3D printing and designing models for 3D printing. A quick Google search led us to long tutorials and complex functionalities that we didn’t need for the design of our educational puzzle. After discussing the problem with friends and colleagues from other faculties, we figured out a workflow for designing a 3D model without CAD software experience. This method focuses on designing as much of the model as possible as a 2D vector graphic and requires minimal work in the 3D software. This way, we could develop our puzzle from scratch. So, besides the actual puzzle itself, we wanted to share a guide for future iGEMers who also have no previous experience with 3D printing but would like to design a simple model for educational or even wet lab purposes. This guide can be found in the first section of the handbook (p. 1-16).

Updated STIR Protocols

Reflexivity is crucial for understanding the societal, ethical, and material dimensions of research, yet it can be challenging to integrate into specific workflows. A key aspect of our Human Practices work was revising the STIR (Socio-Technical Integration Research) protocols to foster deeper reflexivity in a practical and flexible way. Building on iGEM Imperial College’s 2016 use of STIR, we tailored the framework to better suit our contexts—such as research sessions, PI meetings, and stakeholder engagements. Our updated protocols are structured to help future teams apply STIR more effectively within their own internal and engagement practices.

The protocol templates can be found below, and details about how their use impacted our work are present on our Human Practices page.

References

Florea, M., Hagemann, H., Santosa, G., Abbott, J., Micklem, C. N., Spencer-Milnes, X., de Arroyo Garcia, L., Paschou, D., Lazenbatt, C., Kong, D., Chughtai, H., Jensen, K., Freemont, P. S., Kitney, R., Reeve, B., & Ellis, T. (2016). Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain. Proceedings of the National Academy of Sciences, 113(24), E3431–E3440. https://doi.org/10.1073/pnas.1522985113