Difference between revisions of "Part:BBa K731400"

Revision as of 22:15, 6 September 2012

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 iGEM Trento 2012 wiki page.

ATTENTION: When using this part special handling and precaution should be taken, as this enzyme produces H2S. Manipulation of cells producing this part should be done under a chemical hood.

Usage and Biology

CysDes is a unique 45 KDa hemolysin cysteine dependent, that was shown to have also aminostransferase activity. (1, 2) The enzyme catalyze 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.

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 on the Trento 2012 wiki.

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 37C in LB until it was reached an OD of 0.4. The cells were at this point span down and resuspended in an equal volume of MOPS medium and allowed to grow 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). Legend: yellow, MOPS A -IPTG; orange, MOPS A +IPTG, light blue, MOPS B -IPTG; dark blue, MOPS B +IPTG.

FIGURE 2. CysDes toxicity test by serial dilution. Cells were grown under the same conditions described in figure 1. At 4 and 8 hours of induction a 500 ul 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 ul aliquot of each serial dilution was plated on LB agar and placed overnight at 37C. The following day the number of colonies from each plate were 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. Legend: yellow, MOPS A -IPTG; orange, MOPS A +IPTG, light blue, MOPS Bl -IPTG; dark blue, MOPS B +IPTG.

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.

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 as described in REF. Briefly, every hour a 1 mL aliquot of cells was taken and span down. To the supernatant was added 1 ul of a 100mM solution of Bathocuproinedisulfonic reagent and 1 ul of a 1M ascorbate solution and the solution was vortexed. Absorbance was measured at 483 nm. Legend: Panel A, Uninduced cells, Panel B Induced cells, in blue is shown Absorbance at 483 nm and in red is shown optical density.

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 (MODEL and Brand). Measurements were taken 3 times at interval of 2 minutes. A calibration curved 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