Difference between revisions of "Part:BBa K731480"
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'''FIGURE 1.''' NEB10b cells transformed with BBa_K731480 were grown in LB at 37C 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 span down and resuspended in 1.5 mL of PBS. Fluorescence measurements were taken with a Varian Cary Eclipse Spectrophometer using an excitation wavelenght of 464 nm.
 
'''FIGURE 1.''' NEB10b cells transformed with BBa_K731480 were grown in LB at 37C 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 span down and resuspended in 1.5 mL of PBS. Fluorescence measurements were taken with a Varian Cary Eclipse Spectrophometer using an excitation wavelenght of 464 nm.
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'''FIGURE 2.''' Protein expression at different IPTG concentrations
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'''FIGURE 3.'''
  
 
===References===
 
===References===

Revision as of 00:15, 31 August 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.

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 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.

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.


Andrea2.jpg

FIGURE 1. NEB10b cells transformed with BBa_K731480 were grown in LB at 37C 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 span down and resuspended in 1.5 mL of PBS. Fluorescence measurements were taken with a Varian Cary Eclipse Spectrophometer using an excitation wavelenght of 464 nm.



FIGURE 2. Protein expression at different IPTG concentrations


FIGURE 3.

References

1. INFECTION AND IMMUNITY, Nov. 1995, p. 4448–4455
2. Appl Microbiol Biotechnol (2003) 62:239–243

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2460
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1559
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2490