Difference between revisions of "Part:BBa K4011003"
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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 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. | 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== | |
| − | + | ||
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 analysis. The relative concentrations of Trp and 6-X-Trp was calculated for every sample. | 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 analysis. The relative concentrations of Trp and 6-X-Trp was calculated for every sample. | ||
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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. | 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. | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K4011003 SequenceAndFeatures</partinfo> | <partinfo>BBa_K4011003 SequenceAndFeatures</partinfo> | ||
Revision as of 10:10, 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
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 analysis. The relative concentrations of Trp and 6-X-Trp was calculated for every sample.
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 576
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 948
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 424