Difference between revisions of "Part:BBa K731480"

Latest revision as of 21:21, 26 October 2012

IPTG Inducible sfGFP tagged Cysteine desulfhydrase (CysDes)

A sfGFP tagged CysDes inducible by IPTG.

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 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].

For a characterization on the enzymatic activity check also to part BBa_K731400.

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 [http://2012.igem.org/Team:UNITN-Trento wiki].

Usage and Biology

CysDes is a unique 45 KDa hemolysin cysteine dependent, that was shown to have also aminostransferase 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. Protein expression and cell growth upon IPTG induction
NEB10b cells transformed with BBa_K731480 were grown in LB at 37°C until OD of 0.7 and induced with different concentrations of IPTG. After 4 hours a 1.5 mL aliquot was taken from each sample. Cells were spun down and resuspended in 1.5 mL of PBS. Panel A: Fluorescence measurements were taken with a Varian Cary Eclipse Spectrophometer using an excitation wavelenght of 464 nm. Panel B: Optical density at 600 nm.

FIGURE 2. Effects of glycerol and glucose on protein expression levels
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 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. After 4 hours a 1.5 mL aliquot was taken to measure fluorescence intensity. The assay was performed always in the presence of 1 mM L-cysteine and in two different MOPS media. MOPS A: 60 mM glycerol (yellow). MOPS B: 30 mM glucose (blue). The experiment has been performed in triplicate.

FIGURE 3. Effect of cysteine on protein expression levels
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 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. After 4 and 8 hours of induction a 1.5 mL aliquot was taken to measure fluorescence intensity. The assay was performed in the presence and in the absence of 1 mM L-cysteine (cys) and two different media: MOPS A, 60 mM glycerol (yellow), MOPS B, 30 mM glucose (blue).

FIGURE 4. CysDes toxicity test by optical density and serial dilution
Cells were grown under the same conditions described as before. 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 1:10ˆ6. 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 were counted. Conditions used are MOPS with 60 mM glycerol (A) and MOPS with 30 mM glucose (B). All experiments were done in the presence and in the absence of 1 mM cysteine (cys). Panel A: Optical density at different time points in MOPS A (A) and B (B). Colonies Forming Unit (CFU) at 4 hours and 8 hours in MOPS A (C) and MOPS B (D).

References

1. Chu L, Burgum A, Kolodrubetz D, and Holt SC. The 46-kilodalton-hemolysin gene from Treponema denticola encodes a novel hemolysin homologous to aminotransferases. Infect Immun 1995 Nov; 63(11) 4448-55.
2. Awano N, Wada M, Kohdoh A, Oikawa T, Takagi H, and Nakamori S. Effect of cysteine desulfhydrase gene disruption on L-cysteine overproduction in Escherichia coli. Appl Microbiol Biotechnol 2003 Aug; 62(2-3) 239-43.

Sequence and Features