Difference between revisions of "Part:BBa K5226086"

 
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<partinfo>BBa_K5226086 short</partinfo>
 
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<h2>Sequence and Features</h2>
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<partinfo>BBa_K5226086 SequenceAndFeatures</partinfo>
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<body>
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<h2>Introduction</h2>
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<br>
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One of the goals of iGEM SCUT-China-A is to use synthetic biology tools to obtain <i>Halomonas</i> strains that can produce tyrian purple. We chose to introduce four enzymes that is either necessary or beneficial to the production of tyrian purple. There were stth,fre,tnaA and fmo. Because both Stth and TnaA can utilize tryptophan, and tryptophan has a stronger preference for TnaA than for Stth, we introduced the <b>thermalsensitive bio-switch</b> that we built for <i>Halomonas TD</i> to <b>separate the expression</b> of the two enzymes to increase yield.
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<h2>Usage and Biology</h2>
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<br>
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This is a composite part used to convert 6-Br-Trp to 6-Br-indole and further to Tyrian Purple. TnaA is a kind of tryptophanase and this protein catalyzes the conversion of 6-Br-Trp into 6-bromoindole (6-Br-indole). MaFMO is a kind of flavin-containing monooxygenase and the protein catalyzes the conversion 6-Br-indole into 6,6'-dibromoindigo (6BrIG, also known as Tyrian purple). They are fused together with the common rigid linker EAAAKEAAAK. Through introducing thermalsensitive bio-switch into the synthesis pathway of Tyrian purple, we could <b>use temperature to separate the expression of stth and tnaA</b>, thus improve the production of 6-Br-Trp and then the Tyrian purple. Considering its importance and expression intensity, we selected the Mmp1 inducible promoter and set a series of IPTG concentrations during fermentation to induce the most suitable expression intensity for this step.
  
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<h2>Experimental characterisation</h2>
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<h3>growth conditions</h3>
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<html> <img src="https://static.igem.wiki/teams/5226/parts/bba-k5226060-mmp1-am1-c1m-3.jpg" width="700px">
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<h3>Shake flask fermentation</h3>
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<b>Strain preparation</b>
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<h3>experimental design</h3>
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Other variables were the amount of IPTG and tryptophan added. We set two gradients for each of the two variables: IPTG(mg/L)=2,5 and tryptophan(g/L)=0.4,0.8.
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<br>
 +
At 16,24,32h, we took a batch of samples and switched the temperature to 37℃, in case to find a better timing to switch temperature through the yield of 6-Br-Trp.
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<h3>Data Processing and Analysis</h3>
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<h2>References</h2>
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[1]Feifei Li, Que Chen, Huaxiang Deng, Shumei Ye, Ruidong Chen, Jay D. Keasling, Xiaozhou Luo,One-pot selective biosynthesis of Tyrian purple in Escherichia coli,Metabolic Engineering,Volume 81,2024,Pages 100-109.
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<br>
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[2]Athina Vasileiadou, Ioannis Karapanagiotis, Anastasia Zotou,
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Determination of Tyrian purple by high performance liquid chromatography with diode array detection,Journal of Chromatography A,Volume 1448,2016,Pages 67-72.
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<br>
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[3] Li, F., Chen, Q., Deng, H., Ye, S., Chen, R., Keasling, J. D., & Luo, X. (2024). One-pot selective biosynthesis of Tyrian purple in Escherichia coli. Metabolic Engineering, 81, 100–109.
  
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K5226086 SequenceAndFeatures</partinfo>
 
  
  

Revision as of 11:42, 30 September 2024

PRM-ci857-mmp1-fre-stth

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 2844
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1973
    Illegal NgoMIV site found at 2231
    Illegal NgoMIV site found at 2934
    Illegal NgoMIV site found at 3200
    Illegal AgeI site found at 2119
    Illegal AgeI site found at 2460
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1362
    Illegal SapI.rc site found at 1482

Introduction


One of the goals of iGEM SCUT-China-A is to use synthetic biology tools to obtain Halomonas strains that can produce tyrian purple. We chose to introduce four enzymes that is either necessary or beneficial to the production of tyrian purple. There were stth,fre,tnaA and fmo. Because both Stth and TnaA can utilize tryptophan, and tryptophan has a stronger preference for TnaA than for Stth, we introduced the thermalsensitive bio-switch that we built for Halomonas TD to separate the expression of the two enzymes to increase yield.

Usage and Biology


This is a composite part used to convert 6-Br-Trp to 6-Br-indole and further to Tyrian Purple. TnaA is a kind of tryptophanase and this protein catalyzes the conversion of 6-Br-Trp into 6-bromoindole (6-Br-indole). MaFMO is a kind of flavin-containing monooxygenase and the protein catalyzes the conversion 6-Br-indole into 6,6'-dibromoindigo (6BrIG, also known as Tyrian purple). They are fused together with the common rigid linker EAAAKEAAAK. Through introducing thermalsensitive bio-switch into the synthesis pathway of Tyrian purple, we could use temperature to separate the expression of stth and tnaA, thus improve the production of 6-Br-Trp and then the Tyrian purple. Considering its importance and expression intensity, we selected the Mmp1 inducible promoter and set a series of IPTG concentrations during fermentation to induce the most suitable expression intensity for this step.

Experimental characterisation

growth conditions



Shake flask fermentation

Strain preparation


experimental design


Other variables were the amount of IPTG and tryptophan added. We set two gradients for each of the two variables: IPTG(mg/L)=2,5 and tryptophan(g/L)=0.4,0.8.
At 16,24,32h, we took a batch of samples and switched the temperature to 37℃, in case to find a better timing to switch temperature through the yield of 6-Br-Trp.

Data Processing and Analysis

References

[1]Feifei Li, Que Chen, Huaxiang Deng, Shumei Ye, Ruidong Chen, Jay D. Keasling, Xiaozhou Luo,One-pot selective biosynthesis of Tyrian purple in Escherichia coli,Metabolic Engineering,Volume 81,2024,Pages 100-109.
[2]Athina Vasileiadou, Ioannis Karapanagiotis, Anastasia Zotou, Determination of Tyrian purple by high performance liquid chromatography with diode array detection,Journal of Chromatography A,Volume 1448,2016,Pages 67-72.
[3] Li, F., Chen, Q., Deng, H., Ye, S., Chen, R., Keasling, J. D., & Luo, X. (2024). One-pot selective biosynthesis of Tyrian purple in Escherichia coli. Metabolic Engineering, 81, 100–109.