Difference between revisions of "Part:BBa K3034005"

Revision as of 08:45, 21 October 2019

PtisAB

P_tisAB 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.

P_tisAB sequence was derived from Escherichia coli K12 MG1655 and has been codon optimized [1].（GenBank：AE000445.1）

Usage and Biology

The Mechanism of P_tisAB

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 is cleared, whereby the promoter starts and expresses the downstream gene [2].

Fig. 1. Schematic diagram of CIP-induced P_tisAB principle

As the schematic of the piGEM2019-01 shown above(Fig. 1), when P_tisAB senses ciprofloxacin, subsequent genes will express, one of them is GFP. By detecting the fluorescence intensity of GFP, the expression of P_tisAB 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 Fluorescence in unit OD for wild-type Ⅰ E.coli DH5α（a）and E.coli DH5α carrying piGEM2019-01(b). wild-type E.coli DH5αcarries a plasmid which only have AmpR. Cells were exposed to 0 0.1 1 6 10mg/L CIP in exponential phase.Fold induction is GFP unit fluorescence after 2/4/6 h 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 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.

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.

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.

Conclusion

For E.coli DH5α carrying piGEM2019-01, we can see P_tisAB responds differently to CIP at different concentrations, among them, 1mg/L is the most appropriate response concentration for P_tisAB.

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