Difference between revisions of "Part:BBa K731400"

Revision as of 11:59, 29 August 2012

__This part encodes the BBa_K731600 cysteine desulfhydrase (CysDes) from Treponema denticola downstream of a strong expression IPTG inducible cassette 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 pSB4K4. A sfGFP tagged fusion of this part has been deposited as BBa_K731480 and used to test the protein expression levels upon IPTG induction.

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.

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.

Usage and Biology

CysDes is a unique 45 KDa hemolysin 45KDa cysteine dependent, that was shown to have also aminostransferase activity. The enzyme catalyze the degradation of L-cysteine to produce hydrogen sulfide, ammonia and pyruvate. (FIGURE)

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 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 sample 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 and in the absence of 1 mM L-cysteine and in two different MOPS media. MOPS A: xx mM glycerol (colore). MOPS B: xx mM glucose. Each experiment has been performed in triplicate.

FIGURE 2 CysDes toxicity test by serial dilution. Cells were grown under the same conditions described as before. 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 onto LB agar plates and a placed overnight at 37C. The following day the number of colonies from each plate were counted. Conditions used are MOPS with glycerol (colore) and MOPS with glucose (colore). All experiments were done in the presence of 0.1 mM cysteine.

FIGURE 3 H2S production by methylene blue assay. The production of H2S was observed by a colorimetric assay as described by the Keasling group in Appl. Environ. Microbiol., 2000, 4497-502.). 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 XX.

FIGURE 4 Gas Chromatography profile of H2S production. 50 mL of cells were grown as previously described in a sterile bottle with a modified screw cap that allows to connect the bottle directly to the instrument. After 4 hours of induction the

Sequence and Features