Part:BBa_K5226087
PR/PRM -tnaA -fmo
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
https://static.igem.wiki/teams/5226/parts/bba-k5226060-mmp1-am1-c1m-2.jpg
https://static.igem.wiki/teams/5226/parts/bba-k5226060-mmp1-am1-c1m-3.jpg
Shake flask fermentation
Strain preparationexperimental design
Data Processing and Analysis
To ensure the measurement accuracy of the spectrophotometer, we diluted the bacterial solution 5 times and measured OD600.Data Processing and Analysis
The experimental results showed that the introduction of AM1-C1M increased the growth of TD80 by 67.33% under the culture conditions with sodium formate as the sole carbon source, while the introduction of Vib-C1M increased it by 59.67% After importing C1M, the ability of TD80 to assimilate formate was significantly improved. We urgently hope to explore whether overexpression of C2M and C3M will further enhance the ability of TD80 to assimilate formate. For further experiments, please turn to BBa_K5226063.References
[1] Kim S, Lindner S N, Aslan S, et al. Growth of E. coli on formate and methanol via the reductive glycine pathway[J]. Nature chemical biology, 2020, 16(5): 538-545.[2] Yishai O, Bouzon M, Doring V, et al. In vivo assimilation of one-carbon via a synthetic reductive glycine pathway in Escherichia coli[J]. ACS synthetic biology, 2018, 7(9): 2023-2028.
[3] Turlin J, Dronsella B, De Maria A, et al. Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains promotes synthetic formate assimilation[J]. Metabolic Engineering, 2022, 74: 191-205.
[4] Claassens N J, Bordanaba-Florit G, Cotton C A R, et al. Replacing the Calvin cycle with the reductive glycine pathway in Cupriavidus necator[J]. Metabolic Engineering, 2020, 62: 30-41.
[5] Tian J, Deng W, Zhang Z, et al. Discovery and remodeling of Vibrio natriegens as a microbial platform for efficient formic acid biorefinery[J]. Nature Communications, 2023, 14(1): 7758.
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