Difference between revisions of "Part:BBa K3034005"
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====The Mechanism of P<sub>tisAB</sub>==== | ====The Mechanism of P<sub>tisAB</sub>==== | ||
− | Ciprofloxacin can cause the SOS reaction of the bacteria, and the SOS reaction activates the RecA enzyme. When the RecA enzyme is activated, the LexA repressor | + | Ciprofloxacin can cause the SOS reaction of the bacteria, and the SOS reaction activates the RecA enzyme. When the RecA enzyme is activated, the LexA repressor will be cleared, whereby the promoter starts and expresses the downstream gene [2]. |
[[File:T--UESTC-China--Module001加原理.png|800px|thumb|center|'''Fig. 1''' Schematic diagram of CIP-induced P<sub>tisAB</sub> principle]] | [[File:T--UESTC-China--Module001加原理.png|800px|thumb|center|'''Fig. 1''' Schematic diagram of CIP-induced P<sub>tisAB</sub> principle]] | ||
− | As the schematic of the piGEM2019-01 shown above(Fig. 1), when P<sub>tisAB</sub> senses ciprofloxacin, subsequent genes will express, one of them is GFP. By detecting the fluorescence intensity of GFP, the expression of P<sub>tisAB</sub> can be reflected. | + | As the schematic of the piGEM2019-01 shown above(Fig. 1), when P<sub>tisAB</sub> senses ciprofloxacin, subsequent genes will express, and one of them is GFP. By detecting the fluorescence intensity of GFP, the expression of P<sub>tisAB</sub> can be reflected. |
====Detection of Green fluorescence intensity — Methods==== | ====Detection of Green fluorescence intensity — Methods==== | ||
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2. Take 800μL from yesterday's bacteria and prepare a 15mL system (100mL small conical flask), cultivate the bacteria to the logarithmic growth phase(1.5h). | 2. Take 800μL from yesterday's bacteria and prepare a 15mL system (100mL small conical flask), cultivate the bacteria to the logarithmic growth phase(1.5h). | ||
− | 3. | + | 3. Separately add CIP to prepare a system having a CIP concentration of 0, 0.1, 1, 6, 10 mg/L. |
4. Detect OD600 and green fluorescence after 0h, 2h,4h,6h by a multi-function microplate reader. | 4. Detect OD600 and green fluorescence after 0h, 2h,4h,6h by a multi-function microplate reader. | ||
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• OD600 detection method: in 96-well plate, 3 duplicate holes, minus LB blank. | • OD600 detection method: in 96-well plate, 3 duplicate holes, minus LB blank. | ||
− | • Green fluorescence intensity detection method: At each time point, 1.5mL of bacterial liquid was taken in a light-proof tube,centrifuged at 12000rpm for 20 minutes, washed once with distilled water, and resuspended once. Add 96-well plate 200μL, excitation wavelength at 475nm, emission wavelength at 520nm. | + | • Green fluorescence intensity detection method: At each time point, 1.5mL of bacterial liquid was taken in a light-proof tube, centrifuged at 12000rpm for 20 minutes, washed once with distilled water, and resuspended once. Add 96-well plate 200μL, excitation wavelength at 475nm, emission wavelength at 520nm. |
====Detection of Green fluorescence intensity — Results ==== | ====Detection of Green fluorescence intensity — Results ==== |
Revision as of 12:44, 21 October 2019
PtisAB
PtisAB is a promoter which can be induced by ciprofloxacin(CIP) through SOS response, and the expression of it varies with different concentrations of CIP. This part is responsible for starting to express subsequent genes once it sensed CIP.
PtisAB sequence was derived from Escherichia coli K12 MG1655 and has been codon optimized [1].(GenBank:AE000445.1)
Usage and Biology
The Mechanism of PtisAB
Ciprofloxacin can cause the SOS reaction of the bacteria, and the SOS reaction activates the RecA enzyme. When the RecA enzyme is activated, the LexA repressor will be cleared, whereby the promoter starts and expresses the downstream gene [2].
As the schematic of the piGEM2019-01 shown above(Fig. 1), when PtisAB senses ciprofloxacin, subsequent genes will express, and one of them is GFP. By detecting the fluorescence intensity of GFP, the expression of PtisAB can be reflected.
Detection of Green fluorescence intensity — Methods
Fixed strain: The experimental group used E.coli DH5α carrying piGEM2019-01. The control group used wild-type Ⅰ E.coli DH5α.
1. A blank vector and a single clone of E.coli DH5α carrying piGEM2019-01 and wild-type E.coli DH5α were taken from the crossed plates, and 5 mL of LB and 5 μL of ampicillin were added to a 12 mL BD tube, and incubated for 11 hours.
2. Take 800μL from yesterday's bacteria and prepare a 15mL system (100mL small conical flask), cultivate the bacteria to the logarithmic growth phase(1.5h).
3. Separately add CIP to prepare a system having a CIP concentration of 0, 0.1, 1, 6, 10 mg/L.
4. Detect OD600 and green fluorescence after 0h, 2h,4h,6h by a multi-function microplate reader.
• OD600 detection method: in 96-well plate, 3 duplicate holes, minus LB blank.
• Green fluorescence intensity detection method: At each time point, 1.5mL of bacterial liquid was taken in a light-proof tube, centrifuged at 12000rpm for 20 minutes, washed once with distilled water, and resuspended once. Add 96-well plate 200μL, excitation wavelength at 475nm, emission wavelength at 520nm.
Detection of Green fluorescence intensity — Results
![](/wiki/images/7/73/T--UESTC-China--PtisABWT1.png)
Compared E.coli DH5α carrying piGEM2019-01 with wild-type E.coli Ⅰ DH5α, we can see that the fluorescence intensity in E.coli DH5α carrying piGEM2019-01 is significantly stronger than wild-type Ⅰ E.coli DH5α.
To determine that our green fluorescence is produced by ciprofloxacin-induced promoter expression, we performed the same experimental procedure on Wild-type Ⅱ (with an arabinose-inducible promoter) , in order to make sure that other promoters won’t be induced by CIP. Besides, we narrowed the range of concentration gradients to find the most appropriate concentration of ciprofloxacin and the linear relationship between green fluorescence intensity and CIP concentration.
![](/wiki/images/3/3d/T--UESTC-China--PtisAB%E5%B0%8F%E8%8C%83%E5%9B%B4%E6%94%B9.png)
Conclusion
For E.coli DH5α carrying piGEM2019-01, we can see PtisAB responds differently to CIP at different concentrations, among them, 1mg/L is the most appropriate response concentration for PtisAB.
Besides, We can infer the concentration of ciprofloxacin based on the green fluorescence intensity. At a concentration of 0-1 mg/L of ciprofloxacin, its relationship with green fluorescence intensity is basically in accordance with y = 0.3698x + 0.5477. R2 = 0.9721 (y: the green fluorescence intensity; x: the ciprofloxacin concentration).
References
[1]Vogel J, Argaman L, Wagner EG, Altuvia S: The small RNA IstR inhibits synthesis of an SOS-induced toxic peptide. Current biology : CB 2004, 14(24):2271-2276.
[2]Dörr T, Vulić M, Lewis K: Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli. PLoS Biol 2010, 8(2):e1000317-e1000317.
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]