Part:BBa_K2742001
HADase 12
The gene encodes for a Haloacid Dehalogenase (HADase) protein. This protein is capable of degrading chlorinated organic compounds, including dichloroacetate (DCA). The sequence for this DNA was codon-optimized and synthesized by IDT. The synthesized DNA was amplified using gene-specific vectors and inserted into the Nde1/Sal1 site of the pET-28a(+) expression vector using Gibson assembly. The plasmid was transformed into Top10 as the cloning strain and verified using both colony PCR and plasmid restriction digest as can be seen here:http://2018.igem.org/Team:UT-Knoxville/Demonstrate.
To verify protein expression, IPTG-induced protein activation and HPLC analysis were used. Firstly, we created a pre-batch of our K1 DNA vector and a BL21 DE3 control with an empty backbone. The medium we used for our pre-culture was LB broth with Kan-50. The pre-batch grew overnight at 37°C at a RPM 220.
Once the pre-batch was complete, we acquired two 250mL flasks that had been autoclaved and added 50mL of LB and Kan-50 to both flasks. We then inoculated with 1mL (1/50 Dilution) of pre-batch culture. Our cultures incubated for 8 hours at 37°C at 220 RPM. IPTG was added, at a 1mM final concentration, to our culture and grown overnight. The next day we took the culture from the flask and added them into falcon tubes. We made this switch as we needed to centrifuge our cell culture so that we could remove the cells from the medium. After centrifuging, we removed the supernatant and resuspended the cells in Tris-Buffer. We then used a sonication machine to lyse our cells and release our HADase proteins into the supernatant. We then centrifuged cell materials could be removed leaving supernatant (crude cell extract) rich in HADases.
Acquired from a metagenomic dataset of an Austrian wastewater sample, the HADase12 was prepared and tested via HPLC. The absorbance peaks of DCA and Glyoxylate were pre-measured for reference before any samples were measured. We then had timed-interval samples. At time t=0, DCA was first added to the crude cell extract. We tested very frequently since the Km of our protein was unknown. 500uL crude cell extract was used for this experiment.
Our findings on HAD12 showcases the prevalence of dehalogenation in varied environments and provides an impetus for our continued work examining the ecological importance dehalogenation.
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