Part:BBa_K2742000

HADase 1

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. <p>In order to test our first HADase, HADase1, we created three testing groups. For group 1 used 50uL of crude cell extracts to test, group 2 we used 20uL of crude cell extract, and lastly group 3 we added 50uL of control crude cell extract. We added 3.5mM of DCA to each group and quickly took a sample. The results of this test can be observed below in Cell extracts of strain HAD1 + DCA: 20 and 50 uL extracts. Each sample was added to a microcentrifuge tube with 1 uL of sulfuric acid to denature the HADase protein and stop its catalytic activity. For the HPLC, the absorbance peaks of DCA and Glyoxylate were pre-measured for reference before any samples were measured. We then had timed-interval samples, i.e. we took samples at 0, 10, 30, 45, 60, 120, 240, and 360 minutes. 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. With a better understanding of the HADase's substrate binding efficiency, a second test was run with a far higher crude cell volume of 500uL crude cell extract. Clearer data was obtained, demonstrating that our enzyme successfully functions by degrading dichloroacetate. Degradation of DCA by HADase1 overtime in the second trial is depicted below in 500 µl Cell Extracts of Strain HAD1 + DCA . <p> To further demonstrate the efficacy of our HADase1 enzyme and its potential as a more general tool for removing chlorinated solvents, we decide to test the enriched crude extract using monochloroacetate (MCA). As can be seen in 500 µl cell extracts of strain HAD1 + MCA our extract was capable of rapidly breaking down MCA giving us evidence that HADase1 could be used as a general tool for the removal of chlorinated solvents. As the need to remove multiple pollutants is necessary in the real-world applications, this shows a significant step towards being able to develop organisms that can effectively remove pollutants from contaminated water sources.

Image on left

<img src="" align = "left" width = 100%; height=110%; >

The gene encodes for a Haloacid Dehalogenase (HADase) protein. This protein is capable of degrading chlorinated organic compounds, including dichloroacetate (DCA) and monochloroacetate (MCA).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]