Difference between revisions of "Part:BBa K4011003"

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(→‎Usage and Biology)
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Fre-SttH is a fused protein used to halogenate the 6th carbon of the tryptophan in the dye production. Fre-SttH is composed of two separate domains-Fre is from E.coli and SttH is from Streptomyces toxytricini. They are fused by a rigid linker with the protein sequence EAAAKEAAAK.  SttH is a trp-6-haloganese that requires FADH2 as a cofactor to convert trp into 6-X-trp, and is highly insoluble in E. coli. Therefore, Fre, a highly-soluble flavin reductase which reduces FAD to FADH2 from E. coli, is fused with SttH as a N-terminal soluble tag, enabling the protein to become soluble and eliminating the need for costly FADH2 cofactors to be added.
 
Fre-SttH is a fused protein used to halogenate the 6th carbon of the tryptophan in the dye production. Fre-SttH is composed of two separate domains-Fre is from E.coli and SttH is from Streptomyces toxytricini. They are fused by a rigid linker with the protein sequence EAAAKEAAAK.  SttH is a trp-6-haloganese that requires FADH2 as a cofactor to convert trp into 6-X-trp, and is highly insoluble in E. coli. Therefore, Fre, a highly-soluble flavin reductase which reduces FAD to FADH2 from E. coli, is fused with SttH as a N-terminal soluble tag, enabling the protein to become soluble and eliminating the need for costly FADH2 cofactors to be added.
 
 
 
 
−
<br> To express Fre-SttH, we first attempted the T7 system, commonly used in E. coli BL21(DE3). We expressed histag-Fre-SttH using the T7 system with a lacO promoter, but found both its expression and solubility to be fairly low, thus making it unsuitable for our project. 
+
<br>  
 +
To express Fre-SttH, we first attempted the T7 system, commonly used in E. coli BL21(DE3). We expressed histag-Fre-SttH using the T7 system with a lacO promoter, but found both its expression and solubility to be fairly low, thus making it unsuitable for our project. 
 
 
 
 
−
<br> Because we needed a knockout TnaA (TnaA is naturally expressed in E. coli, giving it its characteristic scent) strain to express Fre-SttH so that our natural trp will not be converted to indole without being halogenized by Fre-SttH, we switched from the T7 system to the ptac system. The ptac system, unlike the T7 system which can only be used in E. coli BL21, can be used in all E. coli strains. Our knockout strain was supplied in E. coli DH5α, courtesy of Sha Zhou, in which we constructed ptac-histag-Fre-SttH and ptac-Fre-SttH. 
+
<br>  
 +
Because we needed a knockout TnaA (TnaA is naturally expressed in E. coli, giving it its characteristic scent) strain to express Fre-SttH so that our natural trp will not be converted to indole without being halogenized by Fre-SttH, we switched from the T7 system to the ptac system. The ptac system, unlike the T7 system which can only be used in E. coli BL21, can be used in all E. coli strains. Our knockout strain was supplied in E. coli DH5α, courtesy of Sha Zhou, in which we constructed ptac-histag-Fre-SttH and ptac-Fre-SttH. 
 
 
 
 
−
<br> We then expressed induced expression of both proteins (histag-Fre-SttH and Fre-SttH) and performed SDS-PAGE analysis. Results show that histag-Fre-SttH expression and solubility were poor, but Fre-SttH had extremely high expression and high solubility. Thus, ptac-Fre-SttH in ΔtnaA E. coli DH5α was used for all further experiments. 
+
<br>  
 +
We then expressed induced expression of both proteins (histag-Fre-SttH and Fre-SttH) and performed SDS-PAGE analysis. Results show that histag-Fre-SttH expression and solubility were poor, but Fre-SttH had extremely high expression and high solubility. Thus, ptac-Fre-SttH in ΔtnaA E. coli DH5α was used for all further experiments. 
 
 
 
 
−
<br> In order to quantify our enzymatic rate of Fre-SttH and measure the substrate-product concentrations of Trp and 6-X-Trp, we induced Fre-SttH expression and added Trp and NaCl/NaBr. We then took samples of the culture every 6 hours for 24 hours for HPLC.  
+
<br>  
 +
In order to quantify our enzymatic rate of Fre-SttH and measure the substrate-product concentrations of Trp and 6-X-Trp, we induced Fre-SttH expression and added Trp and NaCl/NaBr. We then took samples of the culture every 6 hours for 24 hours for HPLC.  
  
−
<br> The rate of conversion from Trp + NaCl to 6-Cl-Trp was faster than that of its Br counterpart, reaching around 1.4mM after 24 hours for Cl, compared to 1.0mM for Br. To learn more about how we did the experiment, visit our experiment and measurement pages. From this, we acquired several enzymatic constants, which was vital to our modeling team.  
+
<br>  
 +
The rate of conversion from Trp + NaCl to 6-Cl-Trp was faster than that of its Br counterpart, reaching around 1.4mM after 24 hours for Cl, compared to 1.0mM for Br. To learn more about how we did the experiment, visit our experiment and measurement pages. From this, we acquired several enzymatic constants, which was vital to our modeling team.  
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 10:21, 20 October 2021


Fre-SttH

Fre-SttH is a fused protein used to halogenate the 6th carbon of the tryptophan in the dye production. Fre-SttH is composed of two separate domains, fre and sttH. Fre is from E.coli and SttH is from Streptomyces toxytricini. They are fused by a rigid linker with the protein sequence EAAAKEAAAK. SttH is a trp-6-haloganese that requires FADH2 as a cofactor to convert trp into 6-X-trp, and is highly insoluble in E. coli. Therefore, Fre, a highly-soluble flavin reductase which reduces FAD to FADH2 from E. coli, is fused with SttH as a N-terminal soluble tag, enabling the protein to become soluble and eliminating the need for costly FADH2 cofactors to be added. The fre-sttH in ptac system helps to improve the solubility of it and also allows it to be expressed in any strain of E.coli. The activity and substrate-product concentrations of fre-sttH is measured using HPLC.

Usage and Biology

Fre-SttH is a fused protein used to halogenate the 6th carbon of the tryptophan in the dye production. Fre-SttH is composed of two separate domains-Fre is from E.coli and SttH is from Streptomyces toxytricini. They are fused by a rigid linker with the protein sequence EAAAKEAAAK.  SttH is a trp-6-haloganese that requires FADH2 as a cofactor to convert trp into 6-X-trp, and is highly insoluble in E. coli. Therefore, Fre, a highly-soluble flavin reductase which reduces FAD to FADH2 from E. coli, is fused with SttH as a N-terminal soluble tag, enabling the protein to become soluble and eliminating the need for costly FADH2 cofactors to be added.  
To express Fre-SttH, we first attempted the T7 system, commonly used in E. coli BL21(DE3). We expressed histag-Fre-SttH using the T7 system with a lacO promoter, but found both its expression and solubility to be fairly low, thus making it unsuitable for our project.   
Because we needed a knockout TnaA (TnaA is naturally expressed in E. coli, giving it its characteristic scent) strain to express Fre-SttH so that our natural trp will not be converted to indole without being halogenized by Fre-SttH, we switched from the T7 system to the ptac system. The ptac system, unlike the T7 system which can only be used in E. coli BL21, can be used in all E. coli strains. Our knockout strain was supplied in E. coli DH5α, courtesy of Sha Zhou, in which we constructed ptac-histag-Fre-SttH and ptac-Fre-SttH.   
We then expressed induced expression of both proteins (histag-Fre-SttH and Fre-SttH) and performed SDS-PAGE analysis. Results show that histag-Fre-SttH expression and solubility were poor, but Fre-SttH had extremely high expression and high solubility. Thus, ptac-Fre-SttH in ΔtnaA E. coli DH5α was used for all further experiments.   
In order to quantify our enzymatic rate of Fre-SttH and measure the substrate-product concentrations of Trp and 6-X-Trp, we induced Fre-SttH expression and added Trp and NaCl/NaBr. We then took samples of the culture every 6 hours for 24 hours for HPLC.


The rate of conversion from Trp + NaCl to 6-Cl-Trp was faster than that of its Br counterpart, reaching around 1.4mM after 24 hours for Cl, compared to 1.0mM for Br. To learn more about how we did the experiment, visit our experiment and measurement pages. From this, we acquired several enzymatic constants, which was vital to our modeling team.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 576
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 948
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 424