Part:BBa_K4153004
Added by THINKER_CHINA
Procedures to prove our lysis module using copper-sensitive promoter:
1. Cultivate E. Coli in LB mediums at 37 degrees Celsius and 200 rpm.
2. When OD600 values equals to 0.3, add different concentrations of copper, three times for each group.
3. Measure OD600 values at 0.5h, 1h, 1.5h, 2h, 3h and 4h intervals.
Simialr to the results from the pBad/araC promoter, the graph suggested that the lysis circuit works regularly when the concentration of copper is above 10^-6 mol/L.
Copper promoter +srrz cell lysis gene
Promoter from the copper-sensitive CusR/CusS two component signal system. (E.Coli)
This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in E.coli. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of CusA, CusB, CusC and CusF genes coding the proteins of copper metabolic system.
The SRRz gene codes maybe three protein S,R,Rz.The product of S gene would cause lesions on the cytoplasmic membrane through which the product coded by the R gene escapes to the periplasm and causes murein-degrading, while the Rz gene’s product may be an endopeptidase that can cleave the oligopeptide crosslinks in the peptidoglycan and/or between peptidoglycan and the outer membrane.
Procedures to prove our lysis module using lac promoter:
1. Cultivate E. Coli in LB mediums at 37 degrees Celsius and 200 rpm.
2. When OD600 values equals to 0.4, add different concentrations of copper, three times for each group.
3. Measure OD600 values at 0.5h, 1h, 1.5h, 2h, 3h and 4h intervals.
the graph suggested that the lysis circuit works regularly when the concentration of copper is above 10^-5 mol/L.
Our system is initiated by two components: pBad/araC promoter (BBa_I0500) and copper-sensitive promoter (BBa_I760005). Being two efficient and stable promoters, they induce the expression of lysis genes inserted in the bacterial plasmids productively, guaranteeing the working efficiency of our lysis module.
Procedures to prove our lysis module using pBad/araC promoter:
1. Cultivate E. Coli in LB mediums at 37 degrees Celsius and 200 rpm.
2. When OD600 values equals to 0.3, add different concentrations of arabinose, three times for each group.
3. Measure OD600 values at 0.5h, 1h, 1.5h, 2h, 3h and 4h intervals.
The results suggested that the lysis circuit works regularly when the concentration of arabinose is above 10^-6 mol/L. The rapid decline of OD600 at 10^-5 mol/L indicates lysis of bacterial wall, which proves that our lysis module could function normally and continue to work in a relevant context.
Procedures to prove our lysis module using copper-sensitive promoter:
1. Cultivate E. Coli in LB mediums at 37 degrees Celsius and 200 rpm.
2. When OD600 values equals to 0.3, add different concentrations of copper, three times for each group.
3. Measure OD600 values at 0.5h, 1h, 1.5h, 2h, 3h and 4h intervals.
srrz cell lysis gene
The SRRz gene codes maybe three protein S,R,Rz.The product of S gene would cause lesions on the cytoplasmic membrane through which the product coded by the R gene escapes to the periplasm and causes murein-degrading, while the Rz gene’s product may be an endopeptidase that can cleave the oligopeptide crosslinks in the peptidoglycan and/or between peptidoglycan and the outer membrane.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |