Difference between revisions of "Part:BBa K4765011"

 
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__NOTOC__
 
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<partinfo>BBa_K4765005 short</partinfo>
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<partinfo>BBa_K4765011 short</partinfo>
  
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MysB (O-MT), an O-methyltrans-ferase, converts Demethyl 4-deoxygadusol into 4-deoxygadusol
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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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(4-DG), completing the production of 4-DG, the basic substrate of MAAs.
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__TOC__
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===Introduction===
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MysB (O-MT), an O-methyltrans-ferase, converts Demethyl 4-deoxygadusol into 4-deoxygadusol (4-DG), completing the production of 4-DG, the basic substrate of 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'''
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|}
  
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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_K4273005 BBa_K4273005(NpR5599)] specifically for the ''Escherichia coli'' K12 strain, resulting in the creation of this part. The enzyme MysB catalyzes the second 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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''
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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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|-
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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_K4765005 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K4765011 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K4765005 parameters</partinfo>
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<partinfo>BBa_K4765011 parameters</partinfo>
 
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==References==
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<references />

Latest revision as of 07:11, 12 October 2023


MysB codon optimized

contributed by Fudan iGEM 2023

Introduction

MysB (O-MT), an O-methyltrans-ferase, converts Demethyl 4-deoxygadusol into 4-deoxygadusol (4-DG), completing the production of 4-DG, the basic substrate of MAAs[1].

contributed by Fudan iGEM 2023
Figure 1. The biosynthetic pathway of shinorine, porphyra-334, palythine-Ser, and palythine-Thr

Usage and Biology

We performed codon optimization on BBa_K4273005(NpR5599) specifically for the Escherichia coli K12 strain, resulting in the creation of this part. The enzyme MysB catalyzes the second 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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 532


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.