Regulatory

Part:BBa_K3034005

Designed by: Jiayi Lu 、Yubing Zhou   Group: iGEM19_UESTC-China   (2019-10-02)
Revision as of 12:57, 21 October 2019 by Crisone (Talk | contribs) (→‎Conclusions)

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 capable 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].

Fig. 1 Schematic diagram of CIP-induced PtisAB principle

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

Fig. 2 Fold of Fluorescence Intensity per OD for control (PtisAB-, a) and E.coli DH5α carrying piGEM2019-01 (PtisAB+, b). When cells grow to exponential phase, 0 0.1 1 6 10 mg/L CIP were added into LB to induce the expression of PtisAB. Fold means GFP unit fluorescence after 2/4/6 h of exposure to CIP normalized to initial unit fluorescence. Unit fluorescence is fluorescence intensity per OD. This graph is a representative of two independent experiments with similar results; error bars indicate the standard error.


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.

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(Fig. 3). In this experiment, we choose Wild-typeⅡ as the control (with an arabinose-inducible promoter), since arabinose-inducible promoter won’t be induced by CIP.


Fig. 3 Fluorescence in unit OD for E.coli DH5α carrying piGEM2019-001 and wild-type Ⅱ E.coli DH5α . Wild-type Ⅱ E.coli DH5α 2 carries a plasmid which has AmpR and GFP, and GFP is initiated by the arabinose-induced promoter. Cells were exposed to 0 0.2 0.4 0.6 0.8 1.0 mg/L CIP in exponential phase. Fold means GFP unit fluorescence after 2h of exposure normalized to initial unit fluorescence.（Unit fluorescence=green fluorescence of 1.5mL bacteria/OD600 of 0.2mL bacteria）This graph is a representative of three independent experiments with similar results; error bars indicate the standard error.

Conclusions

For E.coli DH5α carrying piGEM2019-01, we could see PtisAB responds differently to CIP at different concentrations, and comparison shows that 1 mg/L is the most appropriate response concentration for PtisAB.

Besides, we could infer the concentration of ciprofloxacin from the green fluorescence intensity. At a concentration of 0 – 1 mg/L of ciprofloxacin, it followed the formula 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


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]


[edit]
Categories
Parameters
None