Difference between revisions of "Part:BBa K3036003"
 
(One intermediate revision by the same user not shown)
Line 57: Line 57:
 
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. 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%.
+
After correction with OD600, the result shows a remarkable difference in fluorescence intensity between bacteria induced with 0.01% and 0.05% glucose and a glucose concentration higher than 0.05% did not further repress fluorescence intensity, indicating a threshold of rpoH P5 promoter between 0.01% and 0.05%.  
  
 
[[Image:2019 BNU-China BBa K3036003 pic3(2.0).jpg| border | center | 400px]]<br>
 
[[Image:2019 BNU-China BBa K3036003 pic3(2.0).jpg| border | center | 400px]]<br>
  
−
<div class = "center">Figure 1 Florescence intensity corrected ABS 600 over time</div>
+
<div class = "center">Figure 1 Florescence intensity corrected by OD600 over time</div>
  
 
<b><font size="3">Experimental approach</font></b>
 
<b><font size="3">Experimental approach</font></b>
Line 68: Line 68:
 
2. Culture transformed E. coli in 30mL LB-ampicillin (50 ng/µl) at 37˚C, 180rpm in incubator for 12 hours.<br>
 
2. Culture transformed E. coli in 30mL LB-ampicillin (50 ng/µl) at 37˚C, 180rpm in incubator for 12 hours.<br>
 
3. Pipette 1ml culture into centrifuge tube and centrifuge at 4000rpm for 5 minutes. Discard the liquid.<br>
 
3. Pipette 1ml culture into centrifuge tube and centrifuge at 4000rpm for 5 minutes. Discard the liquid.<br>
−
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.<br>
+
4. Resuspend the collected bacteria with LB-ampicillin (50 ng/µl) containing 0.01%, 0.05%, 0.1% and 1% glucose as experimental groups. Resuspend control group with pure LB-ampicillin (50 ng/µl) medium.<br>
 
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.<br>
 
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.<br>
 
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.<br>
 
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.<br>

Latest revision as of 03:01, 22 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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 correction with OD600, the result shows a remarkable difference in fluorescence intensity between bacteria induced with 0.01% and 0.05% glucose and a glucose concentration higher than 0.05% did not further repress fluorescence intensity, indicating a threshold of rpoH P5 promoter between 0.01% and 0.05%.

2019 BNU-China BBa K3036003 pic3(2.0).jpg

Figure 1 Florescence intensity corrected by OD600 over time

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 0.01%, 0.05%, 0.1% and 1% 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