Team:TAS Taipei/Experiment
Summary
Utilizing Escherichia Coli (E. coli) as our chassis, we synthesized a series of chromoprotein-fused proteins designed to bind to heavy metals and pesticides, which are common agricultural residues. The chromoprotein allows for a convenient visible detection of these residues on agricultural produce. The chromoprotein used in all metal detection tests was mRFP which shows a visible red color. The chromoprotein used in all pesticide detection tests was AmilCP which shows a visible blue color.
Goal
Figure 1. Binding to agricultural residues. This graphic represents our project idea.
The pear on the left is contaminated with agricultural residues, and our colored binding
proteins will bind to the residues and leave a patch of color on the pear (right).
Parts Description - Residue Binding ORFs
Below are our open reading frames that code for a variety of agricultural residue-binding proteins/enzymes.
Bacterioferritin: Natural bacteria iron storage protein
MBP: Lead-binding protein derived from PbrR expressed in cytosol
Metallothionein (CRS5): Protein that can tightly chelate metal ions by forming a strong coordination bond
OprF+CBP: Nonspecific membrane porin for small hydrophilic molecules fused with a copper-binding peptide
LC (Leaf Compost) Cutinase: Fungal enzyme that degrades cutin, water-soluble esters and insoluble triglycerides
OpdA: Phosphotriesterase that can detoxify a broad range of organophosphate pesticides (including paraoxon)
DNA Design and Protein Expression
Figure 2. Construct design of colored residue-binding protein.
We obtained the binding protein DNA from the iGEM Distribution Kit: Bacterioferritin, Metallothionein, MBP, OprF, and LC cutinase. We obtained the binding protein OpdA after it was synthesized by Twist Bioscience. For all of the constructs, we inserted an upstream constitutive strong promoter-RBS combination and a downstream color binding protein, followed by a strong double terminator so as to maximize construct and protein expression.
The results of our Coomassie-stained protein gels indicated the presence of the binding proteins and colored proteins. The protein signals suggested that the proteins were expressed separately, rather than fused together. Since the binding proteins themselves were still expressed, these constructs were still used for preliminary assays.
Figure 3. Construct design of colored residue-binding protein with GS linker.
To ensure that the binding protein and colored protein were fused, we inserted either a flexible glycine-serine linker (GS) or a more rigid EAAAK linker between the binding protein and the chromoprotein. For protein purification in downstream applications, we included a hexahistidine tag (6xHis) after the RBS and before the ORF.
These constructs were synthesized by Twist Bioscience (henceforth called Twist constructs). PCR check and Tri-I Biotech sequencing results indicated that we had successfully cloned these constructs.
The results of our Coomassie-stained protein gels indicated the presence of the binding proteins and colored proteins. The protein signals suggested that the proteins were expressed as a single, fused unit. Additionally, the lack of a protein signal at the chromoprotein size further suggested that the proteins were not separate. Thus, the linker sequence successfully fused the binding protein to the chromoprotein.
For more information about characterization of all our parts, including PCR check and SDS-PAGE gels, refer to our Parts page to find the individual Parts Registry pages for our constructs.
Metal-Binding Protein Assay
Method
Our metal-binding parts produce intermembrane proteins expected to increase the cells’ capacity to store heavy metal ions. To test the functionality of our proteins, we detected the difference in the ion storage capacity of construct-expressing cells and negative-control cells. Thus, we had two experimental groups: cells expressing the fusion protein and cells expressing the binding protein only. We had two negative control groups: cells expressing RFP only and cells carrying an ORF with no promoter (ORF only) plasmid.
Figure 4. Metal binding test groups.
In order to measure cell storage capacity, we incubated cells with the targeted heavy metal ions and then measured the absorbance of extracellular metal ions. By the Beer-Lambert law, concentration is directly proportional to absorbance. For the experimental groups, we expected the extracellular solution to have a lower concentration of metal ions and, thus, a lower absorbance as compared to the negative control.
Figure 5. Expected absorbance spectra for metal-binding test.
Metal solutions were prepared by dissolving the appropriate metal ions into distilled water. To optimize the absorbance measurements in the downstream experiment, the wavelength at the peak absorbance of metal solutions were first determined using a spectrophotometer.
Figure 6. Wavelength for peak absorbance of heavy metal ion solutions. We found the
wavelength for the peak absorbance of nickel to be 651.4 nm, lead to be 298.7 nm, iron to be 776.8 nm,
and copper to be 800.7 nm.
We prepared and standardized overnight bacterial cultures to an OD600 of 0.7. Then, we centrifuged the cultures and resuspended the pellet in heavy metal solution. We gently shook the cell-heavy metal mixtures at room temperature for approximately 2 hours. We then spun down the cells to isolate extracellular solution as the supernatant. We measured the peak absorbance of the supernatant using a spectrophotometer blanked with distilled water.
Figure 7. Metal binding test procedure. The pelleted bacteria were resuspended in metal solution.
Cell cultures are centrifuged after incubation with heavy metals, and the supernatants are measured for absorbance.
Results
Our results indicate that there are lower concentrations of metal ions in the extracellular supernatants of cells expressing the fusion protein and the binding protein, as compared to the negative controls. This shows that our fusion and binding proteins are capable of binding to their targeted metal ions, thus increasing the cell’s ability to retain metal ions from their environment.
Figure 8. Metallothionein and Metallothionein-RFP increase cellular retention of nickel ions.
After two hours of shaking incubation with nickel ions, we centrifuged all samples to isolate the extracellular solution.
At 651.4 nm, we observed lower absorbance in extracellular solution of cells expressing Metallothionein and
Metallothionein-RFP compared to the negative control cells.
We repeated the same metal binding test with fusion proteins synthesized by Twist Bioscience, which included a linker sequence to fuse the binding protein to mRFP. We used cells expressing mRFP only as the negative control.
We found that for all of our Twist constructs except MBP, there are lower concentrations of metal ions in the extracellular supernatants of cells expressing the fused proteins, as compared to negative control. These results suggested that our fused proteins are capable of binding to their targeted metal ions, thus increasing the cell’s ability to retain metal ions from their environment.
Figure 9. Twist metal binding fusion proteins (with GS-linker) increase cellular retention of heavy
metal ions. After two hours of shaking incubation with nickel ions, we centrifuged all samples to
isolate the extracellular solution. We observed lower absorbance in the extracellular solution of cells
expressing the fusion proteins compared to the negative control cells.
We did not test our twist MBP-GS-mRFP construct because during our preliminary metal binding test with our cloned MBP and MBP-mRFP constructs, we observed immediate clumping of the cell-lead mixtures, which resulted in discrepancies in our results. The clumping is most likely a sign of bacteria death in the presence of a high concentration of lead.
(Experiments by Allison K, Yasmin L, Vivian W., Anna C. Figures by Yasmin L, Vivian W.)
Pesticide Binding Enzyme Assay: pH Test
Our pesticide-binding construct, LC-Cutinase, produces intermembrane enzymes expected to break malathion into malathion monoacid and malathion diacid. This means that a functional enzyme would produce an acidic degradation product and thus lower the pH of the solution. To prove that malathion degradation decreases pH, we hydrolyzed Malathion under a high pH environment.
Figure 10. Malathion’s natural degradation in H2O decreases pH. After adding
NaOH to increase the pH malathion to different levels, we measured the pH of malathion
over time. We observed that malathion naturally decreases in pH only when it has a high, basic initial pH.
Our results show that, under high pH, malathion degrades and lowers in pH. Thus, malathion does break down into the two acids to release protons and lower the pH.
To test the functionality of our proteins, we compared the difference in pH levels of solutions of construct-expressing cells and negative-control cells. Thus, we had an experimental set: cells expressing the LC-Cutinase-GS-AmilCP protein, and two negative control groups: cells expressing amilCP only and cells carrying an ORF-only plasmid.
Overnight bacterial cultures were prepared and standardized to an OD of around 0.7. Then, the cultures were centrifuged and the pellet was resuspended in 2 mL of 1:1000 diluted malathion solution. The pH of each solution was measured every 5 minutes over 24 hours.
Figure 11. Pesticide-binding PH test procedure. Cell cultures are centrifuged and resuspended in
malathion. The pH was measured over 24 hours.
Results
Preliminary analysis of the time-based data at room temperature suggested that the enzyme was either not functioning correctly due to being very inefficient (see our Modeling page) or not present. Through more research, we discovered that the optimum condition for LC cutinase is at a pH of 8.5 and a temperature of 50°C (Sulaiman, et al., 2011). We tried these experiments at 50°C and even at optimal conditions, we did not see an improvement in function. This led us to suspect that the enzyme was not actually present in the experiment. When we checked for the presence of our protein by SDS-PAGE, we did not see bands at the correct size, suggesting that LC-Cutinase might not have been expressed in the cells.
(Experiments by Allison K., Yasmin L., Anna C.)
Pesticide Binding Protein Assay: Ellman’s Test
The protein OpdA cleaves malathion to expose its sulfhydryl group (Scott et al., 1970). We found a chemical compound, Ellman’s reagent, that reacts to and turns yellow in the presence of thiol groups like sulfhydryl (ThermoFisher, 2011). The amount of sulfhydryl can be quantified by measuring the absorbance at 412.4 nm. We decided to use Ellman’s to determine the functionality of our OpdA constructs. Additionally, the change in color would mimic the original idea of a visual detection of pesticides.
Method
Through literature research, we found that Ellman’s reagent reacts on a 1:1 molar ratio with malathion. Using the molar weight of both compounds, we calculated the concentration and volume ratio needed for Ellman’s. 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.
Figure 12. 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.
Figure 13. 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.
Utilizing the 6xHis tag on our OpdA construct, we purified the proteins using the His GraviTrap kit (GE Healthcare). We confirmed the presence of our purified proteins by SDS-PAGE.
Figure 14. Protein gel of purified OpdA and OpdA-amilCP proteins.
We used malathion + elution buffer (EB) as our control because our experimental sets also have malathion and EB but with the addition of proteins. In order to mitigate the color of our control, we needed to see if the proteins have an additional effect on the yellow color. We found that the more diluted the protein concentration, the fainter the yellow, which meant our purified proteins contributed more to the yellow color. This also gave us an initial confirmation that OpdA functions as expected.
Figure 15. Visual test with Ellman’s Reagent added. Every cuvette has the volume
ratio of 225uL EB/OpdA/OpdA-amilCP + 150uL H2O/mal + 150uL Ellman’s reagent.
The two on the right look the most yellow, which is expected, as they have our
pesticide-binding proteins and malathion.
To fully test the functionality of OpdA, we ran a time trial of the reaction between OpdA and malathion. We expected that over time, the yellow absorbance would go up, as OpdA cleaves more malathion. We blanked the SpectroVis with EB + H2O + Ellman’s, which was clear. We took absorbance data at 2-minute intervals for a total of 10 minutes, and at 20 and 30 minutes. We measured the absorbance at 412nm at each time point, and recorded the data. Initially, we found that the experimental set turned yellow immediately, and its absorbance values either did not increase or even decreased over time, which suggested the enzymes were so efficient in cleaving the malathion that is was difficult for us to detect an increase in absorbance to model enzyme kinetics. This is great for our final product because we were interested in an immediate color detection of contaminants, but it was hard for us to model the enzyme kinetics. Thus, we diluted our original purified proteins by 1:1000 and repeated the procedure.
Figure 16. Ellman’s test procedure for OpdA proteins and malathion. We created
reaction stocks for OpdA + H2O and OpdA + mal, each with a total volume of 3mL.
At each time point, we took out 375uL from the reaction stock, added them to individual
cuvettes, added 150uL Ellman’s, then took absorbance data.
Results
Our data shows that for the 1:1000 set the absorbance at 412nm does increase over time, which shows that as OpdA cleaves
more malathion, more thiol groups are exposed to Ellman's reagent.
Figure 17. Absorbance of 1:1000 OpdA at 412.nm over time. The absorbance of
OpdA + mal at 412.4nm increases slowly over time, which confirms that OpdA cleaves
more malathion over time.
Our modeling further confirms that we successfully purified the OpdA protein and that it functions as expected (refer to our Modeling page for more information)
(Experiments by Anna C., Figures by Yasmin L.)
Lab Notebook
Cloning Cycle for Metal Binding
Cloning Cycle for Pesticide Binding
Daily Activity Log
Metal Binding Test
Modeling
Pesticide Binding Test
Protocols
Prototype
References
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.
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.
