Difference between revisions of "Part:BBa K1972009"

Revision as of 14:59, 25 October 2016

dszBCAD with Lac promoter

We constructed dszBCAD genes with lac promoter(a weaker promoter to aviod the generation of inclusion body) and choose E.coli BL21 as our host cell. When IPTG is added to our system, DszABCD enzymes will be successful expressed and these four enzymes convert dibenzothiophene (DBT) undergoes through three successive oxidation steps and one a hydrolytic step to 2-hydroxybiphenyl (2HBP) by the 4s pathway.

Each single enzyme gene was synthesized by Generay and we constructed them together and suquencing comformed. the sequences can be found in NCBI (GenBank Accession number L37363.1)

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1266
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 488
Illegal BamHI site found at 2070
Illegal XhoI site found at 480
Illegal XhoI site found at 3225
Illegal XhoI site found at 4066
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 195
Illegal NgoMIV site found at 2514
Illegal NgoMIV site found at 2729
Illegal NgoMIV site found at 3033
Illegal NgoMIV site found at 3153
Illegal AgeI site found at 2688
Illegal AgeI site found at 4254
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 2220
Illegal SapI.rc site found at 3537

Our Previous work shows 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 1). Unfortunately, the desulfurization efficiency was still not significantly improved.

Figure 1. Bio-circuit after first optimization

@@ Line 19: / Line 19: @@
 <partinfo>BBa_K1972009 parameters</partinfo>
 <!-- -->
-[[File: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).
-[[File:T--SCUT-China_A--u22.png|||center|]]
-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.
-[[File:T--SCUT-China_A--u8.png|600px||center|]]
-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.
-[[File:T--SCUT-China_A--u18.png|600px||center|]]
-Figure 4. The desulfurization results of Recombinant strain BL21-dszBCAD tested by HPLC
 Our Previous work shows 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 1). Unfortunately, the desulfurization efficiency was still not significantly improved.