Difference between revisions of "Part:BBa K4765014"

 
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<html><img style="float:right;width:128px" src="https://static.igem.wiki/teams/4765/wiki/2023-b-home.png" alt="contributed by Fudan iGEM 2023"></html>
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In the final step, nonheme iron-(II)- and 2oxoglutarate-dependent (Fe/2OG) oxygenase gene MysH
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===Introduction===
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completes the production of palythines from disubstituted MAAs.
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Nonheme iron-(II)- and 2oxoglutarate-dependent (Fe/2OG) oxygenase gene MysH completes the production of palythines from disubstituted MAAs<ref>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.</ref>.
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{|
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| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/zsl/t-fudan-maa-pathway-wyj.png" alt="contributed by Fudan iGEM 2023"></html>
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| '''Figure 1. The biosynthetic pathway of shinorine, porphyra-334, palythine-Ser, and palythine-Thr. 1'''
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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We performed codon optimization on [https://parts.igem.org/Part:BBa_K4273014 BBa_K4273014] 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''.
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===Characterization===
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====Anti-UV Survival Assay====
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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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{|
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| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/results-wyj/uv.jpg" alt="contributed by Fudan iGEM 2023"></html>
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| '''Figure 2. Anti-UV Assay.'''
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''
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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.
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{|
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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>
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| '''Figure 3. Plates displaying transformed ''E. coli'' after anti-UV assay.'''
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4765008 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K4765014 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K4765008 parameters</partinfo>
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<partinfo>BBa_K4765014 parameters</partinfo>
 
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==References==
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<references />

Latest revision as of 12:41, 12 October 2023


MysH codon optimized contributed by Fudan iGEM 2023

Introduction

Nonheme iron-(II)- and 2oxoglutarate-dependent (Fe/2OG) oxygenase gene MysH completes the production of palythines from disubstituted MAAs[1].

contributed by Fudan iGEM 2023
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 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.

contributed by Fudan iGEM 2023
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.

contributed by Fudan iGEM 2023
Figure 3. Plates displaying transformed E. coli after anti-UV assay.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 23
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

  1. ↑ 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.