Difference between revisions of "Part:BBa K4765013"

(→‎Usage and Biology)
(→‎Anti-UV Survival Assay)
 
(4 intermediate revisions by 2 users not shown)
Line 6: Line 6:
  
 
===Introduction===
 
===Introduction===
−
A D-Ala-D-Ala ligaselike enzyme gene MysD converts Mycosporine-glycine(MG) tO MAA analogues like shinorine or porphyra-334 <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>.
+
A D-Ala-D-Ala ligase gene MysD converts Mycosporine-glycine(MG) tO MAA analogues like shinorine or porphyra-334 <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>.
  
 
===Usage and Biology===
 
===Usage and Biology===
−
We performed codon optimization on [https://parts.igem.org/Part:BBa_K4273013 BBa_K4273013(NlmysD) ] specifically for the ''Escherichia coli'' K12 strain, resulting in the creation of this part. The enzyme MysD catalyzes the fourth reaction involved in the biosynthesis of Mycosporine-like amino acids (MAAs) within ''E. coli''.
+
We performed codon optimization on [https://parts.igem.org/Part:BBa_K4273013 BBa_K4273013] specifically for the ''Escherichia coli'' K12 strain, resulting in the creation of this part. The enzyme MysD catalyzes the fourth reaction involved in the biosynthesis of Mycosporine-like amino acids (MAAs) within ''E. coli''.
 
{|
 
{|
 
| <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>
 
| <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>
 
|-
 
|-
−
| '''Figure1 The biosynthetic pathway of shinorine, porphyra-334, palythine-Ser, and palythine-Thr. 1'''
+
| '''Figure 1. The biosynthetic pathway of shinorine, porphyra-334, palythine-Ser, and palythine-Thr. 1'''
 +
|}
 +
 
 +
===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.
 +
 
 +
{|
 +
| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/results-wyj/uv.jpg" alt="contributed by Fudan iGEM 2023"></html>
 +
|-
 +
| '''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.
 +
 
 +
{|
 +
| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/results-wyj/mysverification.png" alt="contributed by Fudan iGEM 2023"></html>
 +
|-
 +
| '''Figure 3. Plates displaying transformed ''E. coli'' after anti-UV assay.'''
 +
 
 
|}
 
|}
  

Latest revision as of 15:19, 12 October 2023


MysD codon optimized contributed by Fudan iGEM 2023

Introduction

A D-Ala-D-Ala ligase gene MysD converts Mycosporine-glycine(MG) tO MAA analogues like shinorine or porphyra-334 [1].

Usage and Biology

We performed codon optimization on BBa_K4273013 specifically for the Escherichia coli K12 strain, resulting in the creation of this part. The enzyme MysD catalyzes the fourth reaction involved in the biosynthesis of Mycosporine-like amino acids (MAAs) within E. coli.

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

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 1003


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.