Our project went through two separate lines of engineering, with each line containing engineering DBTL cycles. The following is a record of the two lines we followed.
We found information regarding the csoR-Based System from a paper [1], which gave the genetic sequence of A. caldus csoR and introduced its role in A. caldus’s copper detoxification system. The paper identified three possible promoters, respectively named PI, PII, and PIII, for csoR. Although the paper concluded that PII is the copper-sensitive promoter, we suspected that PIII could also become copper-sensitive through adequate improvement. Thus we constructed the PIII-csoR-based system and began characterizing it as the first engineering cycle.
We characterized the csoR-Based System by creating transcription units with csoR and PIII. For more information regarding this, please refer to the protocols on our Experiments page.
After testing csoR with 0 µM, 5 µM, 50µM, 0.5 mM, and 5 mM concentrations of copper, we verified that the PIII-csoR system is indeed not copper-sensitive. For more information regarding this, please refer to our Results page.
From the results, we learned that the original version of the PIII-csoR system does not work as expected, and therefore that further work must be done to alter the structure of PIII and allow the system to work properly.
We designed the improvement process of the PIII-csoR system. Since the main problem is with the copper sensitivity of PIII, we decided to improve the PIII-RBS combination via high-throughput experimentation.
Through random mutation and high-throughput experimentation, we eventually constructed 96 variants of PIII-RBS combinations (registered BBa_K4494003-K4494098 with 78 unique sequences in total). All were characterized. To learn more about how the experimentation process was conducted, please refer to the protocols on our Experiments page.
We conducted a thorough analysis of the 96 variants we got. For more information regarding this, please refer to our Results page.
Finally, through the 96 variants, we found several which are proven as functioning.
We planned to construct a benchmark for our project and try to improve an existing part by characterizing and improving a part in the iGEM registry. After identifying several possible candidates, we eventually selected the CusR-Based System because of its proven superiority and rich documentation.
We mutated part BBa_K1980004 and characterized both the original part and the mutants. To learn more about how the experimentation process was conducted, please refer to the protocols on our Experiments page.
The part BBa_K1980004 and its variants were tested with copper concentrations of 0 and 50µm. Eventually, the characterization results showed that none of the distinct variants were copper-sensitive, indicating that our initial goal has failed. However, we successfully produced a very strong promoter (registered BBa_K4494102).
From this experience, we understood how sometimes an unexpected discovery could occur in the process of scientific research. Although we were unable to further explore this new strong promoter due to limitations in time and funding, we hope that our accidental discovery would set out the first step for future explorations.
[1] Hou, S., Tong, Y., Yang, H., & Feng, S. (2021). "Molecular Insights into the Copper-Sensitive Operon Repressor in Acidithiobacillus caldus". Applied and Environmental Microbiology: 87(16).