iGEM 2022 — Team ict-mumbai

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## Arya's Literature Search

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## Karanveer's Literature Search

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## Shaurya's Literature Search

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## Taha's Literature Search

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## Protocol

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## References

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1. Viegas, Sandra C., Patrícia Apura, Esteban Martínez-García, Víctor de Lorenzo, and Cecília M. Arraiano. "Modulating heterologous gene expression with portable mRNA-stabilizing 5′-UTR sequences." ACS Synthetic Biology 7, no. 9 (2018): 2177-2188.
2. Xiao, Jun, Bing Peng, Zhaowei Su, Ankun Liu, Yajing Hu, Christopher T. Nomura, Shouwen Chen, and Qin Wang. "Facilitating protein expression with portable 5′-UTR secondary structures in Bacillus licheniformis." ACS Synthetic Biology 9, no. 5 (2020): 1051-1058.
3. Pesole, Graziano, Flavio Mignone, Carmela Gissi, Giorgio Grillo, Flavio Licciulli, and Sabino Liuni. "Structural and functional features of eukaryotic mRNA untranslated regions." Gene 276, no. 1-2 (2001): 73-81
4. Kozak, Marilyn. "Initiation of translation in prokaryotes and eukaryotes." Gene 234, no. 2 (1999): 187-208.
5. Chiaruttini, Claude, and Maude Guillier. "On the role of mRNA secondary structure in bacterial translation." Wiley Interdisciplinary Reviews: RNA 11, no. 3 (2020): e1579
6. Hansen, Michael J., Li‐How Chen, Mariena LS Fejzo, and Joel G. Beiasco. "The ompA 5′ untranslated region impedes a major pathway for mRNA degradation in Escherichia coli." Molecular microbiology 12, no. 5 (1994): 707-716.
7. Salis, Howard M., Ethan A. Mirsky, and Christopher A. Voigt. "Automated design of synthetic ribosome binding sites to control protein expression." Nature biotechnology 27, no. 10 (2009): 946-950.
8. Cetnar, Daniel P., and Howard M. Salis. "Systematic quantification of sequence and structural determinants controlling mRNA stability in bacterial operons." ACS Synthetic Biology 10, no. 2 (2021): 318-332.
9. Reis, Alexander C., and Howard M. Salis. "An automated model test system for systematic development and improvement of gene expression models." ACS synthetic biology 9, no. 11 (2020): 3145-3156.
10. Salis, Howard M. "The ribosome binding site calculator." In Methods in enzymology, vol. 498, pp. 19-42. Academic Press, 2011.
11. ARNOLD, TODD E., JEANNE YU, and JOEL G. BELASCO. "mRNA stabilization by the ompA 5′ untranslated region: two protective elements hinder distinct pathways for mRNA degradation." Rna 4, no. 3 (1998): 319-330.
12. Nahvi, Ali, Narasimhan Sudarsan, Margaret S. Ebert, Xiang Zou, Kenneth L. Brown, and Ronald R. Breaker. "Genetic control by a metabolite binding mRNA." Chemistry & biology 9, no. 9 (2002): 1043-1049.
13. Balzer Le, Simone, Ingerid Onsager, Jon Andreas Lorentzen, and Rahmi Lale. "Dual UTR-A novel 5′ untranslated region design for synthetic biology applications." Synthetic Biology 5, no. 1 (2020): ysaa006.
14. Jiang, Weining, Li Fang, and Masayori Inouye. "The role of the 5'-end untranslated region of the mRNA for CspA, the major cold-shock protein of Escherichia coli, in cold-shock adaptation." Journal of bacteriology 178, no. 16 (1996): 4919-4925.
15. Bernstein, Jonathan A., Arkady B. Khodursky, Pei-Hsun Lin, Sue Lin-Chao, and Stanley N. Cohen. "Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays." Proceedings of the National Academy of Sciences 99, no. 15 (2002): 9697-9702.
16. De Lay, Nicholas, Daniel J. Schu, and Susan Gottesman. "Bacterial small RNA-based negative regulation: Hfq and its accomplices." Journal of Biological Chemistry 288, no. 12 (2013): 7996-8003.
17. Hui, Monica P., Patricia L. Foley, and Joel G. Belasco. "Messenger RNA degradation in bacterial cells." Annual review of genetics 48 (2014): 537-559.
18. Fröhlich, Kathrin S., and Jörg Vogel. "Activation of gene expression by small RNA." Current opinion in microbiology 12, no. 6 (2009): 674-682.

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Engineering Cycle
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### Design

### Design

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### Build

### Build

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### Test

### Test

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### Learn

### Learn

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### Here we go again Design-Build-Test-Learn

### Design-Build-Test-Learn

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## Protocol:

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We have created a library of 5'UTRs that gives a range of expressions. The key learning was how just nine nucleotides were enough to influence the gene expression, as observed in the case of cspF. We are still trying to interpret the complex structures of the naturally occurring UTRs. Our next experimental design shall be more complex based on our understanding and observations of GC content. We further plan to add a complexity factor to our next set of experiments by looking at RNAse cleavage sites. We have realised that experimental results are quite different from software predictions. This further justifies the need for more work and attention in the area of 5'UTR.

We conducted our experiments in two types of strains: a cloning strain (DH5alpha) and an expression strain (BL21(DE3)), and obtained expression values with a Spearman's rank correlation coefficient of 0.87. Such a high value indicates that the overall expression trend remains the same across these two strains. We also wish to conduct these experiments in wild-type strains like MG1655 and other types of strains so that the data we generate and contribute to the Library is expansive and useful to as many researchers as possible. We further wish to develop correlations based on experimentally validated data and build on our 5'UTR Library. We have completed the second engineering cycle and are now contemplating the design of the third, as described on our engineering cycle pages.

![Image](https://static.igem.wiki/teams/4303/wiki/bl-all-utrs.jpg)

![Image](https://static.igem.wiki/teams/4303/wiki/final-b21-all.jpg)

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