Difference between revisions of "Part:BBa K3722010"
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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.
 
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.
  
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
  
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<Biology
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  SttH can substitute the 6-Br-Trp into a Bromine atom.
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===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.
 
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.
  
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Usage
 
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Here, we use BBa_K3722006 to increase the solubility of SttH as a fusion enzyme. We transformed the composite part BBa_K3722010 into E. coli BL21(DE3) to improve the yield of 6-chloro-Trp.
 
  
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Characterization
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===Usage===
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The proof of expression
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Here we used <partinfo>Bba_K3722006</partinfo> to increase the solubility of SttH in the form of fusion enzyme. We transformed the composite part <partinfo>BBa_K3722010</partinfo> into E.coli BL21(DE3) to improve the yield of 6-chloro-Trp.
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Based on literature review and pre-experiment, we found it was easy to express in the form of inclusion body. So 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.附上蛋白表达图>
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===Characterization===
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The verification of expression
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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.
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        <img src="https://2021.igem.org/wiki/images/a/ad/T--NWU-CHINA-A--part101.png" width="20%" style="float:center">
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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℃)
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===References===
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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
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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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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2384
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1017
    Illegal NgoMIV site found at 1275
    Illegal AgeI site found at 1504
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 480