Difference between revisions of "Part:BBa K1972014"

Revision as of 15:22, 25 October 2016

dszBBAC with tac promoter and dszD with lac promotor induced by IPTG

We constructed another dszB gene with tac promoter and teminator just front DszBACD cassette. 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 with E.coli optimized and we constructed them together and suquencing comformed. the sequences can be found in NCBI (GenBank Accession number L37363.1)

Sequence and Features

Figure 1. Bio-circuit of BBa_K1972014：Final optimized with dszB copy number increased

Figure 2. Bio-circuit of BBa_K1972008: dszB with tac promoter

Finally, we increased DszB copy number to gain higher efficiency of desulfurization (as shown in Figure 1). We also cultured the recombinant strain BL21-dszB (as shown in Figure 2), and add it (0.5 mgprotein/mL) to the recombinant strain BL21-dszBBACD (optimized)

Figure 3. The desulfurization results of Recombinant strain BL21-dszBBACD (optimized) addition of cell extract from the recombinant strain BL21-dszB tested by HPLC The desulfurization efficiency of the recombinant strain BL21-dszBBACD (optimized) addition of cell extract from the recombinant strain BL21-dszB is further improved (as shown in Figure 3). To know our project working under real industrial condition, we used n-dodecane with DBT (final concentration is 1mM) to simulate oil and the ratio of organic mixture and water is 1:9, which was based on our mathematic modeling. We added induced bacteria into the mixed system at an initial A600 of 2.0. After the reaction of 6 hours, we took 1 ml sample which would be tested by HPLC (as shown in Figure 4). The result shows clearly the generation of 2-HBP which means success of our project. Because it’s only a pre-experiment that we didn’t use equipment such as ultrasonator, the result does not show a high-efficiency desulfurization. We will do 2L-system desulfurization experiment in the next step.

Figure 4. The elements of oil phase and aqueous phase after 6h-reaction The result shows significant conclusions which will guide us in the next step: a.The elements of oil will nearly not be metabolized or absorbed by our engineered bacteria in oil-water phrases. b.Our engineered bacteria can convert DBT into 2-HBP successfully in oil-water phrases. c.Very little organic elements will be left in aqueous phase.