Difference between revisions of "Part:BBa K4765014"
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| − | Our experiments demonstrated that introducing three or four of the five enzymes from the MAA biosynthetic pathway into ''E. coli'' did not yield a significant enhancement in the bacterium's UV resistance. We postulate that this lack of effect may arise from two factors: Firstly, the synthetic pathway may not play a pivotal role amidst the numerous routes involved in MAA synthesis. Secondly, to augment MAA levels within E. coli through protein expression in the pathway, additional substrates like ATP and amino acids would likely be required in the reaction. | + | Our experiments demonstrated that introducing three or four of the five enzymes from the MAA biosynthetic pathway into ''E. coli'' did not yield a significant enhancement in the bacterium's UV resistance. We postulate that this lack of effect may arise from two factors: Firstly, the synthetic pathway may not play a pivotal role amidst the numerous routes involved in MAA synthesis. Secondly, to augment MAA levels within ''E. coli'' through protein expression in the pathway, additional substrates like ATP and amino acids would likely be required in the reaction. |
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| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/results-wyj/mysverification.png" alt="contributed by Fudan iGEM 2023"></html> | | <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/results-wyj/mysverification.png" alt="contributed by Fudan iGEM 2023"></html> | ||
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| − | | '''Figure 3. Plates displaying transformed E. coli after anti-UV assay.''' | + | | '''Figure 3. Plates displaying transformed ''E. coli'' after anti-UV assay.''' |
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Revision as of 08:11, 12 October 2023
MysH codon optimized
Contents
Introduction
Nonheme iron-(II)- and 2oxoglutarate-dependent (Fe/2OG) oxygenase gene MysH completes the production of palythines from disubstituted MAAs[1].
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| Figure 1. The biosynthetic pathway of shinorine, porphyra-334, palythine-Ser, and palythine-Thr. 1 |
Usage and Biology
We performed codon optimization on BBa_K4273014(NlmysH) specifically for the Escherichia coli K12 strain, resulting in the creation of this part. The enzyme MysH catalyzes the final reaction involved in the biosynthesis of Mycosporine-like amino acids (MAAs) within E. coli.
Characterization
Anti-UV Survival Assay
We employed the Colony-Forming Unit (CFU) assay. After plasmid transformation and plating, we shielded one/half of the agar plate from UV light using a black cloth, while the other one/half was exposed to UV irradiation (6W power) with wavelengths of 254 nm and 365 nm for 10 seconds.
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| Figure 2. Anti-UV Assay. |
Our experiments demonstrated that introducing three or four of the five enzymes from the MAA biosynthetic pathway into E. coli did not yield a significant enhancement in the bacterium's UV resistance. We postulate that this lack of effect may arise from two factors: Firstly, the synthetic pathway may not play a pivotal role amidst the numerous routes involved in MAA synthesis. Secondly, to augment MAA levels within E. coli through protein expression in the pathway, additional substrates like ATP and amino acids would likely be required in the reaction.
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| Figure 3. Plates displaying transformed E. coli after anti-UV assay. |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 23
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
- ↑ Chen, M., Rubin, G. M., Jiang, G., Raad, Z., & Ding, Y. (2021). Biosynthesis and heterologous production of mycosporine-like amino acid palythines. The Journal of Organic Chemistry, 86(16), 11160–11168.