Difference between revisions of "Part:BBa K3722010"
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:'''Fig.1''' SDS-PAGE analysis of Fre-Link-SttH . Induction condition: LB media, OD600 0.6 ~ 0.8, IPTG 0.1 mM(1:negitive control 2: 9h 37℃ 3:9h 30℃ 4:9h 23℃ 5:9h 17℃ 6:15h 37℃ 7:15h 30℃ 8:15h 23℃ 9: 15h 17℃) | :'''Fig.1''' SDS-PAGE analysis of Fre-Link-SttH . Induction condition: LB media, OD600 0.6 ~ 0.8, IPTG 0.1 mM(1:negitive control 2: 9h 37℃ 3:9h 30℃ 4:9h 23℃ 5:9h 17℃ 6:15h 37℃ 7:15h 30℃ 8:15h 23℃ 9: 15h 17℃) | ||
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===References=== | ===References=== | ||
Revision as of 23:40, 21 October 2021
T7_Promoter-RBS-Fre-Linker-SttH-T7_Terminator
Fre is a unique N-terminal tag that efficiently enhanced SttH activity by increasing solubility as well as cofactor supply, the improvement might be caused by an enhanced electron transfer efficiency due to the prox-imity effect of the fusion of Fre and SttH, and greatly improved the yield and productivity of bromination.
Biology
SttH can substitute the 6-Br-Trp by a Bromine atom. Fre is a NAD(P)H-flavin reductase from E. coli . It is highly expressed in a soluble form in E. coli and can regenerate FADH2 efficiently.
Usage
Here we used BBa_K3722006 to increase the solubility of SttH in the form of fusion enzyme. We transformed the composite part BBa_K3722010 into E.coli BL21(DE3) to improve the yield of 6-chloro-Trp.
Characterization
The verification of expression
Based on literature consulting and pre-experiment, we found it was easy to express in the form of inclusion body. Thus, we induced BBa_K3722006 at different temperatures to find the optimum conditions. We determined the expression level through SDS-PAGE. The experimental results were shown below.
- Fig.1 SDS-PAGE analysis of Fre-Link-SttH . Induction condition: LB media, OD600 0.6 ~ 0.8, IPTG 0.1 mM(1:negitive control 2: 9h 37℃ 3:9h 30℃ 4:9h 23℃ 5:9h 17℃ 6:15h 37℃ 7:15h 30℃ 8:15h 23℃ 9: 15h 17℃)
References
1.Jeongchan Lee Joonwon Kim et al. Production of T yrian purple indigoid dye from tryptophan in Escherichia coli Nature Chemical Biology | VOL 17 | JANUARY 2021 | 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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2384
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1017
Illegal NgoMIV site found at 1275
Illegal AgeI site found at 1504 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 480