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. Protein expression and cell growth upon IPTG induction''' 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. 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. |
| + | <div style="text-align:center;">[[Image:AT1480_2.jpg]]</div><br/> | ||
| − | + | '''FIGURE 2.''' Effect of glycerol and glucose on protein expression levels. | |
| − | '''FIGURE 2.''' | + | |
Revision as of 17:02, 6 September 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.
FIGURE 1. Protein expression and cell growth upon IPTG induction 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. 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. Effect of glycerol and glucose on protein expression levels.
FIGURE 3.
References
1. INFECTION AND IMMUNITY, Nov. 1995, p. 4448–4455
2. Appl Microbiol Biotechnol (2003) 62:239–243
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2460
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1559
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 2490