Difference between revisions of "Part:BBa K4153004"

Revision as of 09:27, 10 October 2022

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.

Figure 1 OD600 values of different concentration of copper at various timed 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.

Figure 2 Comparison of final OD600 values after 4h、10^-6 mol/L、10^-5 mol/L (copper-sensitive promoter)

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.

Figure 1 OD600 values of different concentration of copper at various timed intervals

the graph suggested that the lysis circuit works regularly when the concentration of copper is above 10^-5 mol/L.

Figure 1 Comparison of final OD600 values after 4h、10^-5 mol/L、10^-4 mol/L (copper-sensitive promoter）

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.

Figure 1 OD600 values of different concentration of arabinose at various timed 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.

Figure 2 Comparison of OD values after 4h、10^-5 mol/L、10^-6 mol/L to control group (pBad/araC promoter)

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