Contribution


Add New Documentation to An Existing Part

We believe that CueR (BBa_K1758320) initiated by different promoters can lead to varying outcomes for the entire system. Promoters are regulatory switches for gene expression, determining under what conditions the downstream genes are activated. We selected several different promoters from the E. coli genome and replaced the original J23119 promoter in the dual-plasmid system to examine the effects these changes would have on the regulatory system.

We picked 96 single colonies from the plate into liquid LB medium for overnight cultivation. The next day, after diluting the overnight culture to logarithmic phase, we added 0 µM and 500 µM copper ion solutions and tested their fluorescence signals overnight.

We observed that all the promoters exhibited induction strength, indicating that each promoter had some level of activation. The lowest showed a 5-fold induction strength, while the highest reached 10-fold. We tested the sequences of these promoters using Sanger sequencing, matched them to their corresponding strengths, and established a model between the induction strength and the sequence characteristics.

Parts Registry: BBa_K1758320

iGEM 2024 Squirrel Shenzhen Team Parts Page


New Composite Parts

We integrated two new composite parts, BBa_K5459002 and BBa_K5459001, using data from previous iGEM databases. We proposed a novel CueR-pCueR regulatory system and modification scheme, significantly enhancing the flexibility and efficiency of copper ion homeostasis regulation in E. coli through innovative design and efficient tools. These innovative tools and resources will provide robust support for research and applications in synthetic biology.


Dual-Plasmid Regulatory System: Achieving Greater Flexibility and Precise Control

We optimized the original single-plasmid design to create a more flexible dual-plasmid system. This modular design not only allows precise control of fluorescent protein expression but also enables flexible adjustment of regulatory elements. The advantages of the dual-plasmid system include:

  1. Independent regulatory elements: By separately controlling the genes on the two plasmids, researchers can more precisely adjust various factors in the regulation process, such as adjusting the expression level or regulatory strength of the fluorescent protein.
  2. Modular design: The dual-plasmid system allows researchers to replace or adjust elements on the plasmids as needed, quickly responding to changes in experimental design.
  3. Enhanced flexibility: This separated design makes regulation under different experimental conditions more flexible, improving the system's operability and experimental efficiency.

Discovery That a Single Copy of Endogenous CueR Protein is Sufficient: Simplified Design, Increased Efficiency, and Expansion to Other Endogenous Proteins

Through systematic studies, we discovered that relying solely on endogenous E. coli CueR protein is sufficient to support normal regulatory function, and in some cases, it performs even better. Traditionally, experimental designs often increase CueR expression through additional plasmids. However, after experimental validation, we reached key conclusions:

  1. A single copy is already effective: Experiments show that solely relying on the endogenous CueR protein in E. coli can ensure efficient system operation, and in certain experimental conditions, it exhibits better stability and regulatory effects.
  2. Simplified system design: External CueR plasmids are no longer needed, greatly simplifying the experimental system, reducing plasmid competition, and decreasing the expression burden, making the system more efficient.
  3. Expansion to endogenous protein research: This discovery is not limited to CueR protein; it also opens new avenues for studying other endogenous regulatory proteins in *E. coli*. In future synthetic biology applications, other endogenous proteins could be explored in similar ways, reducing dependence on exogenous plasmids and further simplifying regulatory system design.

This breakthrough lays the foundation for future research on E. coli endogenous proteins, enabling simpler and more efficient regulatory system designs while providing new research directions for the broader field of biological regulation.


High-Throughput Screening Workflow: A Universal Platform for Multiple Proteins

We developed an efficient high-throughput screening workflow, initially used for screening CueR proteins. This workflow not only significantly improved the efficiency of CueR protein screening but also has **versatility**, making it applicable to research on various proteins. Its key features include:

  1. Standardization and easy dissemination: Our workflow is rigorously optimized to ensure high reproducibility in screening experiments. This workflow performs excellently in internal experiments and can also serve as a universal platform available to other research teams, assisting them in protein screening research across different fields.
  2. Applicable to multiple proteins: Although originally designed for CueR, the workflow has been optimized into a universal tool that can be adapted for screening various proteins. Whether regulatory proteins or other functional proteins, this workflow can be quickly adapted for different protein screening through minor modifications.
  3. Automation and efficiency: The workflow is automated, greatly reducing experimental time and minimizing the complexity of manual operations. Through high-throughput processing, large sample sets can be screened at once, accelerating research progress.

New Composite Parts

Through our high-throughput screening workflow, we also generated a **dataset for indirect characterization of CueR expression levels**. This dataset provides valuable information about CueR expression levels for researchers and includes the following features:

  1. Indirect characterization strategy: We used fluorescent proteins as reporter genes to indirectly measure the regulatory activity of different CueR proteins, providing researchers with an efficient method to quickly evaluate CueR regulatory function.
  2. Data sharing: This dataset will be made available to the community, allowing other researchers to access and use the data as needed, driving innovative research across multiple fields.