Difference between revisions of "Part:BBa K731400"

Revision as of 12:24, 26 October 2012

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].

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.

The production of H2S was confirmed in vitro using lysates of cell transformed with pat BBa_K731400. When induced with IPTG the cells produced H2S, depending on cysteine availability. A small amount of H2S was obtained with uninduced cells, due to the basal expression observed ( BBa_K731480 fig.1). Non transformed cells (empty) due not produce any H2S.

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.

H2S production was quantified using a calibration curve built with Na2S. The data show that our part BBa_K731400 under the conditions used for this assay produces 1 mM H2S when cysteine is provided at a concentration of 1mM, thus suggesting that CysDes produce H2S from cysteine in a 1:1 stochiometric ratio.

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.

From the decrease of the free copper concentration in the medium (fig. 5B), we can deduce that copper formed complexes (covellite) with the H2S produced by or cells transformed with BBa_K731400.

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. The concentration of H2S calculated ranged between 20 and 30 ppm, based on a calibration curve done with H2S.

The H2S concentrations we measured with the gas-chromatographer in the described conditions are not dangerous for human health. However, they are the experimental proof that our engineered system perfectly works, and that H2S production could be exploited in many different ways (for example, for the precipitation of metals like cadmium, copper and so on).

Notes

For the characterization of protein expression levels check BBa_K731480.

References

1. INFECTION AND IMMUNITY, Nov. 1995, p. 4448–4455
2. Appl Microbiol Biotechnol (2003) 62:239–243
3. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2000, p. 4497–4502
3. Appl. Environ. Microbiol., 2000, 4497-502

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1560
  
• 1000
  COMPATIBLE WITH RFC[1000]