Difference between revisions of "Part:BBa K3036003"
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<b><font size="3">Properties </font></b> | <b><font size="3">Properties </font></b> | ||
| − | The glucose-sensitivity is validated by observing GFP expression under the control of rpoH P5 promoter induced with different levels of glucose. The results are as shown below. (Fig 1 | + | The glucose-sensitivity is validated by observing GFP expression under the control of rpoH P5 promoter induced with different levels of glucose. The results are as shown below. (Fig 1) |
| − | After corrected with OD600, the fluorescence intensity shows a remarkable decline upon induction. As is shown in Fig. | + | After corrected with OD600, the fluorescence intensity shows a remarkable decline upon induction. As is shown in Fig. 1, the fluorescence intensity of control group remains stable after induction, whereas all three experimental groups show approximately same degree of decline, indicating a glucose regulatory threshold below 0.1%. |
| − | [[Image: | + | [[Image:2019 BNU-China BBa K3036003 pic3(2.0).jpg| border | center | 400px]]<br> |
| − | <div class = "center">Figure 1 | + | <div class = "center">Figure 1 Florescence intensity corrected ABS 600 over time</div> |
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<b><font size="3">Experimental approach</font></b> | <b><font size="3">Experimental approach</font></b> | ||
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[1] H Nagai, R Yano, J W Erickson, T Yura. Transcriptional Regulation of the Heat Shock Regulatory Gene rpoH in Escherichia coli: Involvement of a Novel Catabolite-Sensitive Promoter. Journal of Bacteriology May 1990, 172 (5) 2710-2715 | [1] H Nagai, R Yano, J W Erickson, T Yura. Transcriptional Regulation of the Heat Shock Regulatory Gene rpoH in Escherichia coli: Involvement of a Novel Catabolite-Sensitive Promoter. Journal of Bacteriology May 1990, 172 (5) 2710-2715 | ||
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Revision as of 22:10, 21 October 2019
rpoH P5 promoter
This rpoH P5 promoter is a glucose inhibitory promoter regulated by cAMP, the receptor protein of which is found to be involved in the heat shock regulatory gene rpoH encoding the σ^32 protein in E.coli. This part is used as a digestion sensor to respond to glucose concentration changes, which is major digestive product of starch in small intestine. As a result, we can have our bilateral switch convert with the changing environment in human intestine.
Biology and Usage
As A glucose-sensitive promoter, rpoH P5, which is localized in 110-bp HindIII-EcoRV segment directly upstream of the rpoH coding region, is directly involved in transcription of a heat shock gene rpoH. It is sensitive to glucose repression, and achieves maximum effect in the presence of cAMP-CRP complex. [1]
This part is used as a digestion sensor, with its ability to respond to glucose concentration changes, which is major digestive product of starch in small intestine. On this basis, we confer our microbe colonized in small intestine a trait that inhibits gene expression in the process of digestion.
| rpoH P5 | |
| Function | Glucose inhibitory promotere |
| Use in | Prokaryotes |
| RFC standard | RFC10 compatible |
| Backbone | pSB1C3 |
| Derived from | Escherichia. coli DH5alpha |
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]
Properties
The glucose-sensitivity is validated by observing GFP expression under the control of rpoH P5 promoter induced with different levels of glucose. The results are as shown below. (Fig 1)
After corrected with OD600, the fluorescence intensity shows a remarkable decline upon induction. As is shown in Fig. 1, the fluorescence intensity of control group remains stable after induction, whereas all three experimental groups show approximately same degree of decline, indicating a glucose regulatory threshold below 0.1%.
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Experimental approach
1. Transform the plasmids into E. coli DH5α competent cells.
2. Culture transformed E. coli in 30mL LB-ampicillin (50 ng/µl) at 37˚C, 180rpm in incubator for 12 hours.
3. Pipette 1ml culture into centrifuge tube and centrifuge at 4000rpm for 5 minutes. Discard the liquid.
4. Resuspend the collected bacteria with LB-ampicillin (50 ng/µl) containing 0.1%, 1% and 2% glucose as experimental groups. Resuspend control group with pure LB-ampicillin (50 ng/µl) medium.
5. Both experimental and control groups are divided into three parallel experiments and cultured in 1mL solution at 37˚C for 8 hours and sampled every two hours. Media are changed every hour through centrifugation and resuspension with fresh media to maintain a steady glucose level.
6. Pipette 100ul culture of each group and mix with 100ul LB-ampicillin (50 ng/µl) in 96-well plate with pure LB-ampicillin (50 ng/µl) as blank to measure GFP florescence intensity and OD600 by microplate reader.
7. Three repicas are tested in each group.
8. Obtain and analyze data.
Reference
[1] H Nagai, R Yano, J W Erickson, T Yura. Transcriptional Regulation of the Heat Shock Regulatory Gene rpoH in Escherichia coli: Involvement of a Novel Catabolite-Sensitive Promoter. Journal of Bacteriology May 1990, 172 (5) 2710-2715