Difference between revisions of "Part:BBa K731400"

Revision as of 21:53, 20 September 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.

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.

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 above (figure 3). Panel A: Intensity of Absorbance at 670 nm at different concentration of cysteine. 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 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: Uninduced cells, Panel B: Induced cells. Absorbance at 483 nm is shown in blue, optical density is shown in red.

FIGURE 6. Gas Chromatography profile of H2S production
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. After 4 hours of induction, with 0.1 mM IPTG, the bottle was attached to a portable gas chromatographer (MICROGC A3000 Agilent). Measurements were taken 3 times at intervals of 2 minutes. A calibration curve was done with H2S.

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]