Part:BBa_K2921260
Promoter + RBS + 6xHIS + OpdA + Double Terminator
This construct constitutively expresses OpdA (Basic part: BBa_K215090). According to iGEM09_Washington’s basic part page of BBa_K215090, OpdA is a phosphotriesterase from Agrobacterium radiobacter that can degrade organophosphate pesticides.
Construct Design
This construct was created to constitutively express OpdA. Sequences used for the promoter, RBS, and double terminator came from parts included in the iGEM distribution kit. This construct consists of a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, the protein-coding gene OpdA (Basic part: BBa_K215090), and a double terminator (BBa_B0015) to end transcription. In addition, for downstream applications, we included a hexahistidine tag (6xHis) after the RBS and prior to the OpdA ORF for easy protein purification.
This entire construct was synthesized by Twist Bioscience.
PCR Check Results
The part was confirmed by PCR using the primers VF2 and VR, as well as sequencing by Tri-I Biotech.
We confirmed the size of K2921260 using the primers VF2 and VR, which resulted in the expected size of around 1.5 kb.
Characterization
We used SDS-PAGE to check for OpdA expression in E. coli carrying our construct. Bacterial cultures expressing either OpdA or BBa_K880005 (empty vector) were grown overnight at 37°C, lysed and run on SDS-PAGE gels. OpdA is approximately 37 kDa, and we observed a strong signal at that size in the OpdA lysate sample which was not present in the empty vector sample, suggesting that OpdA is being expressed in the transformed E. coli.
E. coli carrying BBa_K2921260 was lysed and run through a nickel column (GE Healthcare, 11-0033-99) which bound our His-tagged OpdA protein. Bound proteins were eluted with elution buffer and the purified proteins were subject to SDS-PAGE to check the size. We observed a strong signal at 37 kDa, which corresponds to the size of our His-tagged OpdA protein, suggesting that we were able to successfully purify it.
To verify OpdA expression in E. coli, we subjected OpdA lysate (left gel) and OpdA purified protein (right gel) to SDS-PAGE, expecting a signal at around 37 kDa. On the left gel, we saw a strong signal at around 37 kDa in the OpdA lane, but not in the empty lane that was used as a control. On the right gel, we also saw a strong signal at around 37 kDa in the OpdA purified lane.
Functional Assay with Ellman’s Reagent
The protein OpdA cleaves malathion to expose its sulfhydryl group (Scott et al., 1970). Ellman’s reagent reacts to and turns a visible yellow color in the presence of thiol groups like sulfhydryl (ThermoFisher, 2011). The concentration of thiol groups can be quantified by measuring the yellow absorbance at 412 nm on a spectrophotometer.
As a preliminary test, we prepared cell lysate from cells constitutively expressing our OpdA construct. The OpdA cell lysate was then added to either H2O or malathion. Our results showed that only when malathion was added did we see a noticeable increase in yellow color, both by eye and by absorbance at 412 nm.
Visual comparison of OpdA + mal and OpdA + H2O only with Ellman’s added. Addition of malathion with purified OpdA resulted in a darker yellow compared to when water was added.
Absorbance at 412.4nm for OpdA + mal and OpdA + H2O, with Ellman’s added. OpdA + mal has a higher absorbance value, which shows that OpdA cleaves malathion to expose thiol groups, which reacts with Ellman’s to turn more yellow.
In order to test the functionality of OpdA, we conducted preliminary tests to compare OpdA with and without malathion. In every test, the reaction happened in under one minute with OpdA with malathion showing consistently more yellow than the OpdA without malathion. This suggested that the enzyme is very kinetically efficient. Therefore, we diluted the enzyme one to one million with a starting concentration of 2.8 mg/mL (measured by A280). This allowed us to obtain data before all the malathion was converted into products, providing us time point data. By fitting the OpdA results to Michaelis-Menten kinetics, we were able to predict the intensity of the yellow color in different concentrations of malathion given a fixed waiting time.
We added malathion to purified OpdA and we allowed the reaction to proceed for over 10 minutes. We took out an aliquot from the reaction at different time points and added Ellman’s reagent. We collected experimental data on the absorbance of the yellow color from Ellman’s reagent over time when malathion was added in the presence of OpdA and converted the absorbance to the moles of the product produced. We then fit the data to the unknown efficiency of the system (Vm/Km).
Vm/Km= 11.702714287639628 M^-1 s^-1 Standard Error = 2.2977173322822204 R^2 = 0.9296266659850577
References:
Scott, C., Begley, C., Taylor, M. J., Pandey, G., Momiroski, V., Nigel, … Russell, R. J. (1970, January 1). Free-enzyme bioremediation of pesticides: a case study for the enzymatic remediation of organophosphorous insecticide residues. Retrieved October 19, 2019, from https://researchers.mq.edu.au/en/publications/free-enzyme-bioremediation-of-pesticides-a-case-study-for-the-enz.
DTNB (Ellman's Reagent) (5,5-dithio-bis-(2-nitrobenzoic acid). (2011). Retrieved October 19, 2019, from https://www.thermofisher.com/order/catalog/product/22582.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1110
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 207
Illegal AgeI site found at 402
Illegal AgeI site found at 741 - 1000COMPATIBLE WITH RFC[1000]
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