Results

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Construction of Plasmid Vectors for Protein Expression

Using the Plasmid pSR357.24 as a base, we constructed two expression vectors using a restriction digest followed by Gibson assembly. The digest was performed by a double digest with Bsu36I, as well as EcoRI or BseRI, respectively. In one of the plasmids, we cut out an insert consisting of the already present rewired regulator NarL-YdfI, and in the other, we cut out NarL-YdfI along with its associated promoter PydfJ115.

We designed inserts consisting of the rewired regulators EL222-YdfI and NarL-LuxR. EL222-YdfI was inserted into the plasmid with the YdfI promoter PydfJ115 intact, while for NarL-LuxR, the promoter was replaced by pLuxR.

The plasmid backbones without the insert were purified by gel electrophoresis and extracted using a QIAGEN gel extraction kit.

Figure 1: Gel electrophoresis of plasmid backbonesGel electrophoresis of plasmid backbones.

The inserts were manufactured as gBlocks by Integrated DNA Technologies. Full plasmids were assembled using NEB HiFi Gibson assembly and transformed into NEB5α (New England Biolabs) chemically competent cells. Colonies were selected on LB plates containing chloramphenicol.

After one day, colonies were picked and streaked onto induction plates containing chloramphenicol and 10ng/μl tetracycline. EL222 plates were illuminated with 470nm LEDs, and for NarL, sodium nitrate was added for induction. Unfortunately, none of the streaked colonies showed fluorescence under a 470nm light source.

Figure 2: Plates showing colony platingPlates showing colony plating

Project Achievements

Throughout the summer, we worked on designing a system for chimeric regulator construction with the goal of inducing specific gene expression via reporter genes. While we were able to successfully assemble the plasmid and transform it into E.coli, which was evident from the numerous colonies in our cloning strain NEB5alpha, we were not able to observe any reporter gene activity in our experiments, including in the controls that we did not assemble ourselves but had instead ordered from addgene. While this outcome was not what we had hoped for, it provided valuable insights into our experimental setup, assembly methods, and troubleshooting approaches. Below is a summary of both our achievements and the challenges we faced. Our hypothesis for why our induction has failed so far are multiple, and we are currently planning on checking each untill the Jamboree. First, potentially our Tetracycline induction concentation was too low at 10ng/ul. We are planning to induce again at 25ng/ul. Second, we plan to re screen our inserts. Third, we plan to do another gibson assembly, incase something went wrong with the first. And lastly, we are trying to transform the assembled plasmid into a different strain of E.coli to test for induction again. In theory the NarX-NarL system should be active in DH5alpha derived strains, but as cloning strains they are not ideal protein producers.

  • Achievements:
  • Developed a fully functional ClusterControl web tool for predicting regulatory rewiring points.
  • Conducted comprehensive experimental plans, with protocols and workflows established for DNA transformation, plasmid isolation, glycerol stock preparation, induction, and more.
  • Integrated evolutionary analysis, structural analyses and the DDIZ metric into our predictive models, successfully nominating these methods for broader use in regulatory protein studies.
  • Challenges:
  • Failed to induce reporter gene activity in any of our experiments, including control experiments.
  • Learned the importance of testing all experimental components (including positive controls) under real conditions.
  • Observed possible issues with experimental timing, assembly accuracy, and transformation efficiency that we plan to address in future iterations.
  • Recognized that troubleshooting efforts should include a wider range of experimental variables, including cell growth conditions, plasmid stability, and protein expression timing.

Future Plans

Based on the results and challenges faced during this iGEM cycle, we have several future plans to improve and continue our work:

  • Redesigning and troubleshooting the assembly process to ensure greater control over each step, focusing on identifying any potential bottlenecks in the reporter gene expression system.
  • Incorporating additional validation points, including performing quantitative fluorescence measurements over time to detect even minimal reporter gene activity.
  • Further development and application of the ClusterControl tool to explore new regulatory rewiring opportunities and refine predictions based on real experimental results.

Considerations for Replicating the Experiments

For anyone attempting to replicate our experiments, we recommend closely monitoring the following factors:

  • Ensure that all reagents (e.g., competent cells, transformation buffers) are fresh and handled with care to avoid contamination or degradation, especially the tetracycline used for induction. At these concentations even minimal degradation could lead to a loss of induction
  • Use alternative protocols for transformation and plasmid assembly, such as different strains of E. coli or varying inducer concentrations.
  • Pay attention to experimental timing, as issues with incubation periods, heat shock, or growth conditions could impact the final results.
  • Incorporate rigorous controls, both positive and negative, to ensure that the lack of gene expression is due to experimental variables rather than assembly or plasmid stability issues.