Part:BBa_K731400
IPTG inducible Cysteine desulfhydrase (CysDes)
This part encodes a cysteine desulfhydrase (CysDes) from Treponema denticola (BBa_K731600) downstream of a strong expression IPTG inducible cassette (BBa_K731300) in the pSB1C3 backbone. When transformed in E. coli strain NEB10b and induced with IPTG this biobrick produces an enzyme converting L-cysteine into hydrogen sulfide, pyruvate and ammonia.
This part has been successfully operated and characterized both in pSB1C3 and the low copy vector pSB4K5. A sfGFP tagged fusion of this part has also been deposited as BBa_K731480 and used to test protein expression levels upon IPTG induction.
This part was cloned by the iGEM Trento 2012 team for the creation of an aerobically engineered pathway for the removal of the black crust disfiguring marble stones. Further information about this part and its characterization can be found in the [http://2012.igem.org/Team:UNITN-Trento iGEM Trento 2012 wiki page].
This part is also available in the low copy vector pSB4K5 upon request. Contact us at igemtrento[at]gmail.com.
SAFETY NOTES Please note that this part produces hydrogen sulfide, which is toxic if inhaled in high concentrations. Cells handling should be done under a chemical hood. A safety handbook to work with sulfate reducing bacteria is posted in the [http://2012.igem.org/Team:UNITN-Trento iGEM Trento 2012 wiki page].
Contents
Usage and Biology
CysDes is a unique 45 KDa hemolysin cysteine dependent, that was shown to have also aminotransferase activity. (1, 2) The enzyme catalyzes the degradation of L-cysteine to produce hydrogen sulfide, ammonia and pyruvate.
This part produces high levels of CysDes enzyme upon IPTG induction. Protein expression levels have been monitored with the sfGFP tagged composite part BBa_K731480.
Part BBa_K731400 has been fully characterized in pSB1C3 and also in the low copy vector pSB4K5 using E. coli strain NEB10b.
Effect of CysDes on cell growth
FIGURE 1. Growth in different MOPS media
Cell density was measured at different time points to determine the effect of CysDes expression. Cells were grown at 37°C in LB until it was reached an OD of 0.4. The cells were at this point spun down and resuspended in an equal volume of MOPS medium and allowed to grow again to an OD of 0.6. Prior induction the cells were splitted into two samples of equal volume and one of the two samples was induced with 0.1 mM IPTG. Every hour a 1.5 mL aliquot was taken to measure the OD. This assay was performed in the presence of 1 mM L-cysteine and in two different MOPS media: with 60 mM glycerol (A) and with 30 mM glucose (B).
FIGURE 2. CysDes toxicity test by serial dilutions
Cells were grown under the same conditions described in figure 1. At 4 and 8 hours of induction a 500 µl sample was taken from the uninduced and the induced culture and used to make serial dilutions ranging from 1:100 up to 1:10ˆ7. A 200 µl aliquot of each serial dilution was plated on LB agar and placed overnight at 37°C. The following day the number of colonies from each plate was counted. Conditions used are MOPS with 60 mM glycerol (A) and MOPS with 30 mM glucose (B), both in the presence of 0.1 mM cysteine.
Enzymatic activity of CysDes
FIGURE 3. H2S production upon IPTG induction
H2S production was assayed by methylene blue development as described by the Keasling group (3). Briefly, a 5 mL aliquot of cells were resuspended in 300 mM NaCl, 90 mM EDTA, 50 mM Tris-HCl, pH 7.5 and sonicated 3 times for 10 sec in ice. After centrifugation (13000 RPM, 10 min, 4C) 0.1 mM cysteine was added to each supernatant and the samples were placed at 37C for 1 hour. After incubation 0.1 mL of a 0.02M N,N-dimethyl-p-phenylenediamine sulfate solution in 7.2 M HCl and 0.1 mL of a 0.3 M FeCl3 solution in 1.2 M HCl were added to the lysate. Quantification was done with a UV-VIS spectrometer Perkin Elmer lambda 25 at 670 nm. From left to right: cells expressing part BBa_K731400 - IPTG with cysteine, -IPTG without cysteine; + IPTG with cysteine , + IPTG without cysteine; empty cells with cysteine and without cysteine.
FIGURE 4. H2S production as function of cysteine concentration
H2S production was assayed by methylene blue development as described by the Keasling group (3). Conditions used were the same as described in figure 3. Panel A: Intensity of Absorbance at 670 nm at different concentrations of cysteine, as an indication of H2S production. Panel B: Concentration of H2S produced calculated based on a standard curve made with Na2S.
FIGURE 5. H2S production as a function of copper precipitation
Cells were grown in LB in the presence of 2 mM CuSO4. Optical density and free copper concentration left in the media was measured every hour after induction. Free copper concentration was measured by Bathocuproinedisulfonic (BCS) assay. Briefly, every hour a 1 mL aliquot of cells was taken and span down. To the supernatant was added 1 µl of a 100mM solution of Bathocuproinedisulfonic reagent and 1 µl of a 1M ascorbate solution and the solution was vortexed. Absorbance was measured at 483 nm.
Panel A: Uninduced cells, Panel B: Induced cells. Absorbance at 483 nm is shown in blue,
optical density is shown in red.
FIGURE 6. H2S production analyzed by Gas Chromatography.
PANEL A: 50 mL of cells were grown in LB in a 250 mL sterile bottle with a modified screw cap that allows to connect the bottle directly to the instrument. PANEL B: After 4 hours of induction, with 0.1 mM IPTG, the bottle was attached to a portable gas chromatographer (MICROGC A3000 Agilent). PANEL C: H2S formation was qualitatively assessed by exposure to lead acetate test strips for few seconds. PANEL D: Gas Chromatography analysis of NEB10β cells with and without BBa_K731400. Measurements were taken 3 times at intervals of 2 minutes.
Contribution from iGEM17_USTC
The new part we submitted is part BBa_K2242233. Below is the new characterization for this protein Cysteine Desufhydrase. Come to our wiki for more details about this part! iGEM17_USTC.
Cysteine Desulfhydrase is an aminotransferase that converts cysteine into pyruvate, ammonia, and hy- drogen sulfide. Because cysteine desulfhydrase activity is not restricted to anaer- obic conditions, expression of cysteine desulfhydrase in a suitable host could result in aerobic precipitation of cadmium as cadmium sulfide. (Wang, C., Lum, A., Ozuna, S., Clark, D., & Keasling, J. (2001). Aerobic sulfide production and cadmium precipitation by Escherichia coli expressing the Treponema denticola cysteine desulfhydrase gene. Applied microbiology and biotechnology, 56(3-4), 425-430.) In our project, we need to use CdS nanoparticles to generate electrons utilizing light energy. So we need to find a way to produce CdS nanoparticles on the membrane of our engineered E.coli. The easiest way is to precipitate those nanoparticles directly, using the sulfide ions the bacteria produce and the cadmium ion we add into the reaction system. We introduce this enzyme to produce sulfide ion in the cytoplasm of our E.coli efficiently.
Introduction
This year, we improved the characterization of a part encoding the cysteine desulfhydrase(BBa_K731400) from team iGEM12_TRENTO. We found out a new function of the part, to generate sulfur ions from cysteine to precipitate the cadmium ions. In another word, this protein can increase the bacteria's resistance to a certain concentration of cadmium ions and solve the pollution of cadmium ions. With this part, we can successfully precipitate CdS nanoparticles on the surface of our bacteria.
The reason we chose this part for contribution is that we found out that many characterizations should be added if we want to have a thorough understanding of this part. For example, although it can precipitate cadmium ions to increase the resistance, but what is the maximum concentration of cadmium ions it can take? We did a growth curve test to find it out. Besides, we also used Transmission Electron Microscopy(TEM) to confirm that the precipitations are form on the surface on the bacteria because it needs to attach to the bacteria to function in our project.
1.CysDes-pLuxR-pSB1C3 construction and transformation
We obtained the sequence of CysDes gene from Genebank and synthesized this gene from IDT. We inserted this gene to plasmid pSB1C3 with promoter pLuxR on it which was provided by iGEM headquarters. The sequence of gene CysDes was validated with DNA sequencing by Sangon. We transformed this plasmid plays (one contains gene CysDes and promoter pLuxR) into strain BL21. Then we picked some colonies for cultivation and confirmed the transformation result by PCR (shown in Figure 1). From the result of electrophoresis, we confirmed the transformation of pLCys was success.
Figure 1. Electrophoresis result of bacterial PCR (positive result: Cys+pLuxR-1 Cys+pLuxR-2 negative result:Cys+pLuxR-3 Cys+pLuxR-4)
2.Expression of CysDes
We inoculate 2 mL overnight culture to 200 mL LB media (1mM cysteine, 30mM glucose and 10mM HEPES are included) and cultivate for 2h at 37 ˚C. When OD600 reaches 0.4-0.6, add AHL to final concentration of 250nM. After 3h cultivation, collect the bacteria by centrifugation. Then extract the raw enzyme of CysDes by ultrasonication. We run SDS-PAGE of samples of raw enzyme, cell content obtained by 100 ˚C heating and wild type (shown in Figure 2). The protein CysDes is about 46kDa, we can find obvious bands at the about position of 45kDa which are unique to lanes of cell contents after induction and raw enzyme compared with the wild type. Although there are proteins of similar molecular weight in wild type, darker bands in experiment group meaning a high amount of proteins could prove the existence of high amount of CysDes. From the result of SDS-PAGE, we could confirm the expression of CysDes.
Figure 2. SDS-PAGE of cell lysate
3. Growth curve of E.coli(BL21) under different concentration of Cd2+
In our project, we added Cd2+, which is toxic to bacteria, to our media to generate CdS nanoparticles under the catalysis of our engineered E.coli. Considering that CysDes catalyzes the reduction of cysteine and Cd2+ is transformed to CdS precipitation to some extent, the existence of CysDes can strengthen the resistance to Cd2+. But the substrate of CysDes, cysteine, is a kind of necessary amino acids for bacterial growth. So additional cysteine may affect the metabolic pathway which may lead to the depression of bacteria's growth.
To figure out the impact of different concentration of Cd2+ and CysDes on the growth ofE.coli and determine the appropriate concentration of Cd2+ for growth, we measure OD600 as a data for bacteria concentration at various conditions and time respectively. Then we draw the scatter graph and fit the growth curve with smooth line to show the tendency of growth (shown in Figure 3).
Figure 3. Growth curves of BL21 under different concentration of Cd2+
According to the graph of growth curve, we can reach these conclusions:
(a) Adding of Cd2+ to media impedes the growth of wild type E.coli. But after 18h, wild type bacteria grow in low concentration of Cd2+ media (lower than 0.2mM) will reach the same platform stage as the group of media without Cd2+. Higher concentration of Cd2+(over 0.4mM) will limit the platform stage to a lower OD600.
(b) The metabolism of cysteine by CysDes slows down the growth of E.coli and delays the start of exponential stage compared to the wild type. But BL21 expressing CysDes can still reach the platform stage after 18h in the nearly same concentrations as the wild type.
(c) Adding of Cd2+ to media can also obstacle the growth of E.coli expressing CysDes and delay the start of exponential stage. But the change of Cd2+ concentration has no obvious effect on growth curve and cells expressing CysDes grow under 0.4mM Cd2+ can reach a higher concentration approximate to the group of 0.1mM and 0.2mM Cd2+ than the wild type.
(d) The expression of CysDes does strengthen the E.coli ‘s resistance of Cd2+ toxicity, but also slow the growth of E.coli to some extent. We can supplement the media with appropriate amount of cysteine to reduce the negative impact caused by the expression of CysDes.
4. Enzymatic activity analysis of CysDes in vitro
To analysis the enzyme activity of CysDes, we choose to detect the concentration of S2- which is reduced from cysteine under the catalysis of CysDes. Because of lacking appropriate purifying methods, we just analyze the activity of raw enzyme obtained via bacteria lysis. According to the method described in L.Chu et al. of hydrogen sulfide detection, we first cultivate the 1mL mixture of cysteine, PBS buffer and raw enzyme for 2h at 37 ˚C. Sulfide formation was determined by adding 0.1 ml of 0.02 M N,N-dimethyl-p-phenylenediamine sulfate in 7.2 N HCl and 0.1 ml of 0.3 M FeCl3 in 1.2 N HCl to the reaction tubes. The absorbance at 650 nm was determined after color development for 20 min at 20°C. Sulfide concentration is determined from the standard curve of Na2S.
From figure 4, the concentration of S2- in the group of CysDes is higher than the wild type which proves that CysDes promotes the reduction of cysteine to S2- with good enzymatic activity certainly.
Under the catalysis of same amount of raw enzyme, we measure the production of S2- with various concentration of cysteine after 2h at 37˚C (shown in Figure 5). The value of OD650 has approximate linear relationship with the concentration of cysteine which means CysDes almost catalyzes the reduction of all cysteine to S2- and CysDes functions well.
Figure 4. CysDes enzymatic activity
(20mM cysteine)
Figure 5. S2- production as function of cysteine concentration under catalysis of CysDes
5. Transmission electron microscopy image of CdS nanoparticles on bacteria
From the above, we can see that this enzyme CysDes has strong enzyme activity to generate S2- ion from cysteine. However, in this photocatalyst system, the duty of this enzyme does NOT just stop here. It needs to precipitate CdS nanoparticles with additional Cd2+ ions in the system. In the toxicity text section, we can see that strain expressing CysDes had a better resistance than wild type strain. This primarily proves the assumption that this enzyme can enhance the bacteria’s resistance to Cd2+ ions by precipitating them.
To valid this assumption further more, we used Transmission electron microscopy (TEM) to take a closer look to the bacteria. With this, we can valid the existence of CdS precipitance with our own eyes!
Here, we need to express our most sincere gratitude to those teachers in Center for Integrative Imaging(Hefei National Laboratory for Physical Sciences at the Microscale). They helped us to process those samples for TEM.
We prepared four samples—the strain expressing CysDes with and without additional Cd2+ in it and the same for the wild type strain. Here are the results for these four samples.
Figure 6. TEM results
(from 1 to 5: WT, WT+Cd2+, CysDes, CysDes+Cd2+,CysDes+Cd2+)
As you can see in these five pictures, compared with other samples, group 4 and 5 had more precipitations on the surface of the bacteria significantly. Although we have followed every step in the protocol from other papers, our bacteria did NOT have so much precipitation as them. It may result from the fact that there are too many varieties in this experiments. So we could not repeat their results. However, these pictures still provide us with a solid evidence for our assumption! This enzyme can produce S2-, which can precipitate Cd2+ to form CdS nanoparticles for our final goal——utilizing light energy to generate more electrons!
6. Effect of CdS nanoparticles to cathode-current
To see whether CdS nanoparticles can increase the cathode-current as we expected, we added CdS quantum dots kindly provided by team ShanghaiTech into the culture when we were forming bio-film onto the surface of a graphite electrode. Theoretically, CdS quantum dots would be attached to the surface of the bacteria as the bio-film was formed.
Figure 6. Preparation for bio-film
Figure 7. The way we gave light to the reaction system
Then, we put the cathode running and monitored the current. As you can see in figure 8, the strain pMC, which were co-expressing Mtr CAB and Ccm A-H, had a stronger cathode current than the WT strain before the light was given, which perfectly repeated the result we have done in the conduction system section. After the current was stable, we began to give light to the system. The light’s wave length is 455 nm and the source is a LED light bought from an online shop. The strain pMC with CdS quantum dots on it responded to the light stimulate. It had a stronger current than it was before the light was given. However, those strains without CdS quantum dots on it did NOT respond to light stimulate. Especially, for the pMC group without CdS quantum dots on it, it did NOT have any current change after we give light to the system, which exclude the possibility that the current change was resulted from the Mtr CAB proteins or the Ccm A-H protein. Moreover, after we stopped the light, the current got back to the level it was before we gave the light.
Figure 8. Cathode current
This strongly proved the assumption we had that the CdS can increase cathode current, which means that CdS quantum dots can speed up the electrons transfer process, pumping more electrons from the electrode to the bacteria in the same time utilizing light energy. This may results from the CdS quantum dots’ property as a semi-conductor. In the design part about this photosynthesis system, we have a detailed introduction about light-catalyze of semi-conductor. With light energy, we can active the electrons to the conduction band which also create a hole spontaneously. Then the hole will be filled up with the electrons from the electrode. So the cathode current will increased.
In a word, with this outcome, we can safely conclude that CdS quantum dots can increase the cathode current with its semi-conductor property. So, with this photosynthesis system, we can further increase the speed of the electron transfer process which leads to the improvement of synthesis efficiency.
Reference:
[1] Chu, L., Ebersole, J. L., Kurzban, G. P., & Holt, S. C. (1997). Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities. Infection and immunity, 65(8), 3231-3238.[2] Wang, C., Lum, A., Ozuna, S., Clark, D., & Keasling, J. (2001). Aerobic sulfide production and cadmium precipitation by Escherichia coli expressing the Treponema denticola cysteine desulfhydrase gene. Applied microbiology and biotechnology, 56(3-4), 425-430.
[3] Sakimoto, K. K., Wong, A. B., & Yang, P. (2016). Self-photosensitization of nonphotosynthetic bacteria for solar-to-chemical production. Science, 351(6268), 74-77.
Notes
For the characterization of protein expression levels check BBa_K731480.
This part was used together with part BBa_K731030 to engineer E.coli to aerobically reduce sulfate. More info about this pathway can be found on the Trento 2012 wiki page.
References
<biblio>
- Chu pmid=7591084
- Awano pmid=12883870
- Wang pmid=11010904
</biblio>
Sequence and Features
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- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1560
- 1000COMPATIBLE WITH RFC[1000]
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