Engineering Cycles
Characterization of metal binding proteins
Resolving mutated deletions
Gradient toxicity assays
Spytag - Spycatcher
Engineering cycles summary
Synthetic biology, often referred to as bioengineering, applies engineering principles to the development of biological systems, ensuring they perform the desired function. This approach mirrors the method engineers use to develop products—through an iterative process of design, testing, and refinement. In synthetic biology, this method is known as the Engineering Cycle, and it is repeated until the biological system behaves as intended.
The Engineering Cycle consists of four distinct phases: (1) Design, (2) Build, (3) Test, and (4) Learn (Fig.1). Each phase brings us closer to achieving a biological system capable of solving real-world challenges.
Throughout the execution of our project, we adhered to a structured workflow based on the Engineering Cycle to guide each phase of development (Fig.1). Whenever we faced an obstacle, we analyzed it thoroughly, redesigned components where necessary, and iterated on our workflow to build solutions that met the evolving requirements. By progressing through the design, build, test, and learn phases, we were able to refine our project in a logical and methodical manner. Testing yielded valuable insights, which we integrated back into the design, constantly optimizing our biological system to overcome any hurdles. In this way, the Engineering Cycle was not just a theoretical framework but a dynamic process that empowered us to adapt, troubleshoot, and improve as we progressed.
Throughout our project, we identified several key engineering cycles that were critical in achieving our milestones (Fig.2):
- Choice of metal binding proteins and improving their binding properties.
- Cloning and expression of metal-binding constructs – Inserting the peptides into bacterial strains.
- Development of colorimetric and toxicity assays – Optimizing assays to test the metal accumulation efficiency.
- Polymerization of Spytag and Spycatcher
Figure 2: Overview of the 4 engineering cycles and how they interact.
The identified key engineering cycles are closely interconnected, The first interaction occurs between Cycle 1 and Cycle 2. In the build phase of Cycle 1, mutations were encountered during cloning, which Cycle 2 focuses on resolving. The final learnings of cycle 2 was brought back to complete the cloning in the build phase of Cycle 1.
The second interaction is between Cycle 1 and Cycle 3. When testing the cloned bacteria for metal-binding activity in Cycle 1, improvements were needed in the gradient assay, this was the baseline of Cycle 3. The testing phase in Cycle 1 influenced the design of Cycle 3’s experiments. The final insights from Cycle 3 are then applied back to refine the testing phase of Cycle 1.
There is no direct interaction between Cycle 4 and the previous cycles, as it followed an independent pathway. The diagram (Fig.2) visually represents these interactions, showing how the cycles interrelate to refine the project’s outcomes at each phase.
Cycle 1 - Characterization of Metal Binding Proteins
In this cycle, we focus on bridging computational modeling (Dry lab) and experimental validation (Wet lab) within our project. By creating a feedback loop, we allow computational predictions to inform our experiments, while the outcomes from these experiments refine our future computational analyses. This integrative approach enables us to iteratively optimize our systems and enhance the performance of our metal-binding peptides.
Cycle 1.1
Click a section to learn more! In this cycle, the Test and Learn sections are the same
Cycle 1.2
Click a section to learn more! In this cycle, the Redesign and Build sections are the same!
Cycle 2 - Resolving Mutated Deletions
During the construction of our protein expression systems, we aimed to clone several genes of interest into a pBAD plasmid. However, during this process, we encountered unexpected mutations resulting from blunt-end ligation. These mutations affected two distinct regions: for short peptides expression vectors, mutations were found within the short peptide gene itself, while for larger proteins expression vectors, the mutations occurred in the linker region between the target protein and the sfGFP tag .
These errors posed significant challenges, as they caused frameshift mutations, leading to out-of-frame translation. This cycle focuses on resolving these point mutations and restoring correct in-frame translation for functional protein expression.
Cycle 2.1
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Cycle 2.2
Click a section to learn more!In this cycle the Redesign and Build sections are the same!
Cycle 3 - Gradient toxicity assays
To assess whether metal-binding proteins can enhance the survival rate of E. coli under metal stress, we designed an experiment using a gradient agar plate with increasing concentrations of metal ions. The rationale behind this approach was to expose E. coli to progressively harsher metal conditions, thereby allowing us to observe any potential protective effects conferred by the expression of metal-binding proteins. By comparing the growth and survival of E. coli strains with and without these proteins, we aimed to determine their effectiveness in metal tolerance, providing valuable insights into their functional role in metal binding and detoxification.
Cycle 3.1
Click a section to learn more!In this cycle the Design and Build sections will be the same!
Cycle 3.2
Click a section to learn more!In this cycle the Redesign and Build sections will be the same!
Cycle 4 - Spytag - Spycatcher
Cycle 4
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References
[1] Trehan, A., Kiełbus, M., Czapinski, J. et al. REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering. Sci Rep 6, 19121 (2016). https://doi.org/10.1038/srep19121
[2] Gahlot, D.K., Taheri, N., Mahato, D.R. et al. Bioengineering of non-pathogenic Escherichia coli to enrich for accumulation of environmental copper. Sci Rep 10, 20327 (2020). https://doi.org/10.1038/s41598-020-76178-z