Part:BBa_K2512008
vgb promoter+T7RNA polymerase+T7 promoter+GFP
This part is made up of four parts: vgb promoter, T7RNA polymerase T7 promoter and GFP. Vgb promoter is induced under micro-aerobic condition. When the promoter is activated, T7RNA polymerase expresses. Then the T7RNA polymerase will bind to T7 promoter, which will be reported by GFP.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3515
Usage and Biology
This part is improved by the part BBa_K1555003 , which contains a vgb promoter and a GFP. We added a T7RNA polymerase and a T7promoter to the part BBa_K1555003. So compared with BBa_K1555003, the part BBa_K2512008 had a stronger signal generated by GFP.
Host organism
This plasmid is used in E.coli DH5α.
Functional analysis
1. Transform into competent cells(E. coli DH5α). Take several 50mL centrifuge tubes and add 15mL LB medium, 15 μL chloramphenicol.
2. In the centrifuge tubes numbered 1-6, 100μL DH5α were inoculated and sealed with a lid. 7-12 of the centrifuge tubes were inoculated with 100μL strain, but unsealed(use a gas-permeable membrane).
3. Take a sample every two hours and store on the ice. Oxygen concentration was measured by an oxygen electrode. (each sampling and measuring operation takes about 5 minutes).
4. all samples were added to a 96 well plate, and the corresponding OD600 value and fluorescence intensity value were read out by plate reader.
Result
The part BBa_K2512008 also had a nickname pXS-Oxy4. So we use Oxy4 to refer to BBa_K2512008.
The Results were also shown in our wiki: http://2017.igem.org/Team:Worldshaper-XSHS
Figure 1. The growth of E. coli DH5 α under different conditions
In unsealed condition, the growth condition was much better.
Figure 2. Comparison of dissolved oxygen concentration in different conditions
We set the oxygen concentration in the air to 100%, and the concentration of dissolved oxygen in the medium was detected by an oxygen electrode. After four hours, the oxygen content dropped below 5 percent.The unsealed group fell faster.
Figure 3 fluorescence intensity analysis at the excitation wavelength of 475nm
The fluorescence intensity of each group was measured at the excitation wavelength of 475nm(figure 3). The fluorescence intensity of unsealed group is stronger.
Figure 4. Fluorescence intensity of Oxy4 strain in sealed culture
Figure 5. Fluorescence intensity of Oxy4 strain in unsealed culture
We analyzed the results after correction with the initial OD600, and plotted the correlation between the ajusted value and oxygen concentrations (figure 4, figure 5).In unsealed condition, the DH5αcould reach a lower oxygen concentration and emit a stronger fluorescence.
Comparison between BBa_K1555003 and BBa_K2512008
In our project, the part BBa_K2512008 had a nickname pXS-Oxy4, and the part BBa_K1555003 had a nickname pXS-Oxy1. So we use Oxy4 to refer to BBa_K2512008 and use Oxy1 to refer to BBa_K1555003.
The Results were also shown in our wiki: http://2017.igem.org/Team:Worldshaper-XSHS
Figure 5. fluorescence expression of Oxy1 and Oxy4 at different oxygen concentrations
In our model, Oxy4 is better than Oxy1. For details, please refer to our wiki:http://2017.igem.org/Team:Worldshaper-XSHS
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