Difference between revisions of "Part:BBa K4011003"

Revision as of 11:24, 21 October 2021

Fre-SttH

Fre-SttH is a fusion protein used to halogenate the 6th carbon of tryptophan (Trp) in the dye production of 6, 6’dibromoindigo (tyrian purple) and 6, 6’dichloroindigo (tyrian red), with the help of another fusion protein: TnaA-FMO. Fre-SttH is composed of two separate domains: Fre and SttH, fused together with a rigid linker. This is a part in a part collection where we enable the production of indigo, tyrian purple, and related dyes from tryptophan in E. coli.

The part collection includes: Parts expressing Fre-SttH to convert Trp to 6-X-Trp. BBa_K4011003 and BBa_K4011012 . Parts expressing fusion protein TnaA-FMO to convert 6-X-Trp into indigoid dyes. BBa_K4011004 BBa_K4011005 BBa_K4011013 BBa_K4011014 BBa_K4011015 and BBa_K4011019 .

Our part collection can be used to help and inspire future teams to design and perfect different indigoid dye production pathways in E. coli, adding to the collection.

Usage and Biology

Fre-SttH is a fusion 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 and halogen ions 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. We express Fre-SttH in ptac system and measured its enzymatic activity using HPLC (Lee et al, 2021).

Characterization

SDS-PAGE

We expressed Fre-SttH under T7 promoter in E. coli BL21(DE3) (Fig. 1A). SDS-PAGE of the sample showed decent Fre-SttH expression but suboptimal water solubility, since the supernatant sample has less intense target band compared to the whole cell sample (Fig. 1B).

For producing tyrian purple, Fre-SttH and TnaA has to be in two different strains, so we used a ΔTnaA E. coli strain, courtesy of Sha Zhou. However, our ΔTnaA E. coli was supplied as E. coli DH5α, which was incompatible with the T7 promoter.

Because of the need for a ΔTnaA E. coli strain, we decided to switch from the T7 system to the E. coli DH5α compatible ptac system. We constructed two ptac plasmids, ptac-Fre-SttH and ptac-histag-Fre-SttH, and transformed them into E. coli DH5α ΔTnaA (Fig. 1C).

We then induced expression of both proteins and performed SDS-PAGE. Results show that histag-Fre-SttH expression and solubility were poor, but Fre-SttH had extremely high expression and solubility (Fig. 1D). Thus, ptac-Fre-SttH in E. coli DH5α ΔTnaA was used for all further experiments.

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

@@ Line 23: / Line 23: @@
 ==Characterization==
 ===SDS-PAGE===
-We expressed Fre-SttH under T7 promoter in E. coli BL21(DE3) (Fig. 1A). SDS-PAGE of the sample showed decent Fre-SttH expression but suboptimal water solubility, since the supernatant sample has less intense target band compared to the whole cell sample (Fig. 1B).
+We expressed Fre-SttH under T7 promoter in <i>E. coli BL21(DE3)</i> (Fig. 1A). SDS-PAGE of the sample showed decent Fre-SttH expression but suboptimal water solubility, since the supernatant sample has less intense target band compared to the whole cell sample (Fig. 1B).
-<br>For producing tyrian purple, Fre-SttH and TnaA has to be in two different strains, so we used a ΔTnaA E. coli strain, courtesy of Sha Zhou. However, our ΔTnaA E. coli was supplied as E. coli DH5α, which was incompatible with the T7 promoter.
+<br>For producing tyrian purple, Fre-SttH and TnaA has to be in two different strains, so we used a <i>ΔTnaA E. coli</i> strain, courtesy of Sha Zhou. However, our <i>ΔTnaA E. coli</i> was supplied as <i>E. coli DH5α</i>, which was incompatible with the T7 promoter.
-<br>Because of the need for a ΔTnaA E. coli strain, we decided to switch from the T7 system to the E. coli DH5α compatible ptac system. We constructed two ptac plasmids, ptac-Fre-SttH and ptac-histag-Fre-SttH, and transformed them into E. coli DH5α ΔTnaA (Fig. 1C).
+<br>Because of the need for a <i>ΔTnaA E. coli</i> strain, we decided to switch from the T7 system to the <i>E. coli DH5α</i> compatible ptac system. We constructed two ptac plasmids, ptac-Fre-SttH and ptac-histag-Fre-SttH, and transformed them into <i>E. coli DH5α ΔTnaA</i> (Fig. 1C).
-<br>We then induced expression of both proteins and performed SDS-PAGE. Results show that histag-Fre-SttH expression and solubility were poor, but Fre-SttH had extremely high expression and solubility (Fig. 1D). Thus, ptac-Fre-SttH in E. coli DH5α ΔTnaA was used for all further experiments.
+<br>We then induced expression of both proteins and performed SDS-PAGE. Results show that histag-Fre-SttH expression and solubility were poor, but Fre-SttH had extremely high expression and solubility (Fig. 1D). Thus, ptac-Fre-SttH in <i>E. coli DH5α ΔTnaA</i> was used for all further experiments.
 <span class='h3bb'>Sequence and Features</span>