Difference between revisions of "Part:BBa K1420006"

Latest revision as of 02:14, 18 October 2014

phsABC Generator

Overview and Molecular Function

The phsABC genes from Salmonella enterica serovar Typhimurium LT2 encode thiosulfate reductase, which catalyzes the stoichiometric production of hydrogen sulfide and sulfite from thiosulfate for heavy metal removal by precipitation. The phsABC operon encodes three open reading frames (ORFs), designated phsA, phsB, and phsC. Based on sequence homology to formate dehydrogenase-N, it is predicted that thiosulfate reductase behaves in a similar fashion.¹ The PhsA subunit is predicted to be a peripheral membrane protein active site bis(molybdopterin guanine dinucleotide) molybdenum (MGD) cofactor.¹ PhsC is an integral membrane protein that anchors the other two subunits to the membrane, and contains the site for menaquinol oxidation and two heme cofactors located at opposite sides of the membrane.¹ PhsB is predicted to possess four iron-sulfur centers that transfer electrons between PhsC and PhsA.¹

We sought to both improve and characterize phsABC (Part:BBa_K393000), originally added by the 2010 Yale iGEM team, by incorporating a modified lac promoter that enables constitutive expression rather than IPTG induction thus make it more applicable to scale-up remediation efforts. We also improved the characterization of their part by testing its application for biological precipitation of iron and cadmium in addition to their copper testing to add to the functionality of the part. Information on improvements were added to the experience page.

Mechanism

The reduction of thiosulfate catalyzed by thiosulfate reductase is highly endergonic and must be linked to an exergonic process in order to operate in the desired direction.¹ The process consumes the proton motive force (PMF) as electrons are moved from the cytoplasmic side from the site of menaquinone (MK) oxidation and across the membrane by means of the hemes in the membrane subunit (PhsC) and four iron-sulfur centers in PhsB to the periplasmic side of the membrane, where thiosulfate is reduced (Figure 1).¹

Figure 1. Reaction catalyzed by thiosulfate reductase. The process utilizes the proton motive force of electrons moving from the cytoplasm across to the periplasmic side of the membrane where thiosulfate is reduced to a hydrogen sulfide ion and hydrogen sulfite. (Figure adapted from "Thiosulfate Reduction in Salmonella enterica is Driven by the Proton Motive Force." ¹)

phsABC Expression Modifications

phsABC in plasmid pSB74 was obtained through Addgene from Dr. Jay Keasling's laboratory at University of California, Berkeley. It was shown to have the highest catalytic activity in the IPTG-inducible plasmid pSB74 (Table 1).² The phsABC part was initially added to the registry by the Yale 2010 iGEM Team (Part:BBa_K393000) (inducible by IPTG) to deposit copper sulfide in a specified geometry. We sought to both improve and characterize the part for future utilization in our filtration device. We chose to improve the phsABC biological system by incorporating a modified lac promoter that enables constitutive expression rather than IPTG induction thus make it more applicable to scale-up remediation efforts (Figure 2). We also improved the characterization of their part by testing its application for biological precipitation of iron and cadmium in addition to their copper testing to add to the functionality of the part.

Table 1. phsABC catalytic activity in various plasmids in E. coli. (Table adapted from "Engineering hydrogen sulfide production and cadmium removal by expression of the thiosulfate reductase gene (phsABC) from Salmonella enterica serovar typhimurium in Escherichia coli." ²)

Figure 2. Modified construct containing phsABC. The phsABC operon was amplified from the pSB74 plasmid and inserted into pSB1C3 (shipping vector) and the pBBRBB plasmid (for characterization) with the novel addition of a constitutive promoter.

Experimental Results

In order to measure thiosulfate reducing activity of phsABC of NaS₂O₃ to H₂S, the operon was first inserted into the pBBRBB vector with a constitutive P_lac promoter and transformed into E. coli K12. As a negative control, pBBRBB::gfp was tested under the same conditions. The pBBRBB::phsABC K12 and pBBRBB::gfp K12 cells were grown in three test tubes each containing heavy metal tryptone medium as well as 3mM NaS₂O₃. A third set of test tubes were set up with the same contents except without cells as an additional negative control. After 24 hours of incubation, The exact amount of H₂S present in each of three different sets of tubes was then measured using a hydrogen sulfide assay designed by J.D. Cline in 1968 to determine hydrogen sulfide concentrations in natural waters.² For the protocol of this assay, click Cline Reaction Assay Protocol. This consisted of adding 1x or 0.5x 30μL of Cline's Reagent (2g Diamine + 3g FeCl₃ in 50mL of 50% cool HCl) to 270μL of sample. The results were tested against a known standard curve of various Na₂S concentrations. Each sample was allowed 20 minutes for the color to develop before being diluted 1:10 with water for testing (Figure 3). The plate was then read at 670nm with the numerical results displayed in Figure 4. The bar graph shows that the sulfide concentration was considerable higher for the cultures containing pBBRBB:phsABC (380.1 μM ± 13.5) compared to the pBBRBB::gfp negative control (118.9μM ± 1.1). These results are in line with those seen by the Keasling lab.² Following sulfide measurements, cadmium chloride was added (200 μM), and cells were allowed to incubate without shaking at 37C overnight. Cells were pelleted to look for a color change indicating precipitation of CdS (Figure 7). Cell pellets for K12 expressing phsABS were yellow/brown indicating precipitation of CdS while the vector control cells (pBBRBB::gfp) remained white.

Figure 3. 1:10 dilution of cell samples in Cline's Reagent used for hydrogen sulfide assays. The samples in box 1 contain the Na₂S standards (left to right, 1mM, 0.5mM, 0.25mM, 0.125mM, 0.0613mM, and 0.025mM), box 3 contains pBBRBB:GFP K12 cells, while box 3 contains pBBRBB:phsABC K12 cells. Boxes 4, 5, and 6 are the same as boxes 1, 2, and 3 aside from the fact that 0.5x Cline's Reagent was used as opposed to 1x Cline's Reagent that was used in box 1, 2, and 3. Box 7 contains the abiotic sample (left) and water (right) in 1x Cline's reagent. Samples from box 7 are the same as box 8 aside from using 0.5x Cline's Reagent for the latter.

Figure 4. Hydrogen sulfide assay results for pBBRBB:phsABC K12 cells compared to controls. The production of sulfide in pBBRBB:phsABC K12 cells is over three times the amount produced by the pBBRBB:GFP K12 cells, confirming the thiosulfate reductive function of the phsABC gene.

A second set of experiments was also conducted with pBBRBB:phsABC K12, pBBRBB::GFP K12, and an abiotic control grown in heavy metal tryptone medium, 3mM NaS₂O₃tubes, and 2.5mM Fe(II)Cl₂. Since the phsABC gene is responsible reducing thiosulfate, NaS₂O₃ would be converted to H₂S, which will further react with Fe(II)Cl₂ to produce FeS, a black precipitate. After a 24 hour incubation period, the cell cultures appeared as displayed in Figure 5. The pBBRBB:phsABC K12 cells were the only ones seen to produce FeS, the black precipitate seen in the figure, confirming the role of the phsABC gene in reducing thiosulfate. To affirm that this reaction is also successful under non-enclosed systems, the same sets of samples were also tested on 0.2% plates containing 3mM NaS₂O₃tubes and 2.5mM Fe(II)Cl₂. The results after 24 hours of incubation were similar to the experiments conducted in test tubes (Figure 6).

Figure 5. phsABC activity in an enclosed system. pBBRBB:phsABC K12 and pBBRBB::gfp K12 cells were incubated for 24 hours in heavy metal tryptone medium, 3mM NaS₂O₃tubes, and 2.5mM Fe(II)Cl₂. A test tube without cells (abiotic) was also incubated as a negative control. The production of FeS was only seen in the K12 cells that contained pBBRBB:phsABC. This is due to the fact that phsABC gene reduces the NaS₂O₃ to H₂S, which will further react with Fe(II)Cl₂ to produce FeS, the black precipitate seen in the image.

Figure 6. phsABC activity in an open system. A similar experiment as the one described in Figure 5 was conducted, except that 0.2% agar was used as opposed to liquid media to simulate an open environment. The results on the agar plates were similar to ones seen in Figure 5 with FeS only seen in the K12 cells that contained pBBRBB:phsABC.

Figure 7. Precipitation of Cadmium as CdS. The dark yellow/brown color of the cell pellet (top tube) is indicative of CdS formation in E. coli K12 cells expressing pBBRBB::phsABC versus the normal white cell pellet color seen in E. coli K12 cells with pBBRBB::gfp (bottom tube).

References

1. L. Stoffels et al (2011). "Thiosulfate Reduction in Salmonella enterica is Driven by the Proton Motive Force." Journal of Bacteriology 194(2): 475-485.

2. S. W. Bang et al (2000). "Engineering hydrogen sulfide production and cadmium removal by expression of the thiosulfate reductase gene (phsABC) from Salmonella enterica serovar typhimurium in Escherichia coli." Appl Environ Microbiol. 66(9):3939-44.

3. W. V. Sobczak (2005). "Lindeman's Trophoic-Dynamic Aspect of Ecology: 'Will you still need me when I'm 64?.'"Limnology and Oceanography Bulletin 14(3): 53-67.

Sequence and Features