4 enzyme genes was systhsized respectively and we construted them to one cassette.
We constructed dszBCAD genes with T7 promoter 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.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
T7 promoter (strong promoter from T7 bacteriophage)
T7 promoter is very specific promoter which is transcribed only by specific T7 RNA polymerase. Usually this promoter is used in expression systems where T7 promoter is cotransfected with T7 RNA polymerase. That ensures strong transcription of desired genes.
This single monooxygenase gene was synthesized and the sequence was found in NCBI (GenBank Accession number L37363.1)
DszA is a monoxygenase which can convert DBTsulfone (DBTO2) to 2-(2-hydroxybiphenyl) sulfinate (HBPS) during 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 DszA require reducing equivalents (FMNH2) supplied by a flavinreductase (DszD) while working.
The desulfinase was found in Rhodococcus erythropolis IGTS8 and the gene was synthesized and the sequence was found in NCBI (GenBank Accession number L37363.1).
DszB is a a desulfinase which can convert 2-(2-hydroxybiphenyl) sulfinate (HBPS) to 2-hydroxybiphenyl (2HBP) during 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).
This desulfinase can not work without cell system.
The monooxygenase was found in Rhodococcus erythropolis IGTS8 and the gene was synthesized and the sequence was found in NCBI (GenBank Accession number L37363.1).
DszC is a monoxygenase which can not only convert dibenzothiophene (DBT) to DBT-sulfoxide (DBTO) but also convert DBT-sulfoxide (DBTO) to DBTsulfone (DBTO2)during 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 DszC require reducing equivalents (FMNH2) supplied by a flavinreductase (DszD) while working.
DszC require reducing equivalents (FMNH2) supplied by a flavinreductase (DszD) while working.
This flavinreductase was found in Rhodococcus erythropolis IGTS8 and the gene was synthesized and the sequence was found in NCBI (GenBank Accession number AF048979.1).
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.
This flavinreductase can be repaced by other flavinreductases from other bacteria.
Conversion of the iGEM parts registry to SBOL2.1