Difference between revisions of "Part:BBa K1972003"

 
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<partinfo>BBa_K1972003 parameters</partinfo>
 
<partinfo>BBa_K1972003 parameters</partinfo>
 
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[[File:T--SCUT-China_A--u0.png]]
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Figure 1. Bio-circuit after first optimization
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The native dszABC operon was rearranged and the promoter was replaced in order to avoid overlapping genes, increase the expression of the dsz genes, especially dszB, which encoded the rate-limiting enzyme of the 4S-pathway, and relieve inhibition. Besides, a synthetic dszD cassette which was not linked to the dszABC genes in engineered bacteria IGTS8 was also constructed (Figure 1).
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[[File:T--SCUT-China_A--u22.png|||center|]] 
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Figure 2. DNA gel electrophoresis of constructed dsz cassette（lane 1~4）
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The plasmid that can express T7 RNA polymerase under the induction of IPTG and the plasmid that includes four DSZ genes under T7 promoter were successfully constructed and transformed to BL21. Subsequently, the expression of four DSZ genes was detected by SDS-PAGE. As shown in Figure 3, the four enzymes were expressed in the engineered strain.
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[[File:T--SCUT-China_A--u8.png|600px||center|]] 
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Figure 3. SDS-PAGE analysis of DSZ genes expression
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Control: BL21; 1, 2: Recombinant strain BL21-dszBCAD   
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The desulfurization activity of the recombinant strain BL21-dszBCAD was further measured by chromogenic reaction.
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[[File:T--SCUT-China_A--u18.png|600px||center|]] 
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Figure 4. The desulfurization results of Recombinant strain BL21-dszBCAD tested by HPLC
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The result showed the generation of 2-HBP was successful. However, the desulfurization efficiency of the recombinant strain BL21-dszBCAD showed no significant difference compared with that of IGTS8. This might be due to the high promoter activity of T7 promoter. The excessively strong activity of T7 promoter could result in lots of inclusion body, affecting the desulfurization efficiency of the recombinant strain. In order to solve the formation of inclusion body, the T7 promoter was replaced with Lac promoter (as shown in Figure 4). Unfortunately, the desulfurization efficiency was still not significantly improved.

Revision as of 14:52, 25 October 2016


dszD (a flavinreductase)

DszA, DszB, DszC and dszD are required by the 4S pathway, in which dibenzothiophene (DBT) undergoes three successive oxidation steps and one a hydrolytic step leading to the formation of 2-hydroxybiphenyl (2HBP). And three successive oxidation steps require reducing equivalents (FMNH2) supplied by a flavinreductase (DszD) while working.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 225
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 413
  • 1000
    COMPATIBLE WITH RFC[1000]


T--SCUT-China A--u0.png Figure 1. Bio-circuit after first optimization


The native dszABC operon was rearranged and the promoter was replaced in order to avoid overlapping genes, increase the expression of the dsz genes, especially dszB, which encoded the rate-limiting enzyme of the 4S-pathway, and relieve inhibition. Besides, a synthetic dszD cassette which was not linked to the dszABC genes in engineered bacteria IGTS8 was also constructed (Figure 1).


T--SCUT-China A--u22.png

Figure 2. DNA gel electrophoresis of constructed dsz cassette（lane 1~4）


The plasmid that can express T7 RNA polymerase under the induction of IPTG and the plasmid that includes four DSZ genes under T7 promoter were successfully constructed and transformed to BL21. Subsequently, the expression of four DSZ genes was detected by SDS-PAGE. As shown in Figure 3, the four enzymes were expressed in the engineered strain.


T--SCUT-China A--u8.png

Figure 3. SDS-PAGE analysis of DSZ genes expression Control: BL21; 1, 2: Recombinant strain BL21-dszBCAD


The desulfurization activity of the recombinant strain BL21-dszBCAD was further measured by chromogenic reaction.


T--SCUT-China A--u18.png

Figure 4. The desulfurization results of Recombinant strain BL21-dszBCAD tested by HPLC


The result showed the generation of 2-HBP was successful. However, the desulfurization efficiency of the recombinant strain BL21-dszBCAD showed no significant difference compared with that of IGTS8. This might be due to the high promoter activity of T7 promoter. The excessively strong activity of T7 promoter could result in lots of inclusion body, affecting the desulfurization efficiency of the recombinant strain. In order to solve the formation of inclusion body, the T7 promoter was replaced with Lac promoter (as shown in Figure 4). Unfortunately, the desulfurization efficiency was still not significantly improved.