Difference between revisions of "Part:BBa K5226087"

Revision as of 05:45, 30 September 2024

PR/PRM -tnaA -fmo

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 1960
Illegal PstI site found at 724
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 1960
Illegal PstI site found at 724
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 1960
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 1960
Illegal PstI site found at 724
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 1960
Illegal PstI site found at 724
Illegal NgoMIV site found at 1912
Illegal AgeI site found at 1347
Illegal AgeI site found at 2056
Illegal AgeI site found at 2386
1000
COMPATIBLE WITH RFC[1000]

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]Lee, J., Kim, J., Song, J. E., Song, W.-S., Kim, E.-J., Kim, Y.-G., Jeong, H.-J., Kim, H. R., Choi, K.-Y., & Kim, B.-G. (2021). Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nature Chemical Biology, 17(1), 104–112.
[2] Zeng, J., Zhan, J. Characterization of a tryptophan 6-halogenase from Streptomyces toxytricini . Biotechnol Lett 33, 1607–1613 (2011). https://doi.org/10.1007/s10529-011-0595-7

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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</b> 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.
+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.
 <h2>Experimental characterisation</h2>
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 <h3>Shake flask fermentation</h3>
 <b>Strain preparation</b>
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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.
 <br>