Part:BBa_K5226087
PR/PRM -tnaA -fmo
Contents
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1960
Illegal PstI site found at 724 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1960
Illegal PstI site found at 724 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1960
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1960
Illegal PstI site found at 724 - 25INCOMPATIBLE 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 - 1000COMPATIBLE 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]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.
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