Difference between revisions of "Part:BBa K3034005"

Latest revision as of 02:22, 22 October 2019

PtisAB

P_tisAB is a promoter which can be induced by ciprofloxacin(CIP) through SOS response, and the promoter activity of it varies with different concentrations of CIP. This part is capable 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.（GenBank：AE000445.1, MG1655 complete genome 383127...385344）

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 will be cleared, whereby the promoter starts and expresses the downstream genes [1].

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, and one of them is GFP. By detecting the fluorescence intensity of GFP, the promoter activity 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 and wild-type Ⅱ 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 (P_tisAB-, a) and E.coli DH5α carrying piGEM2019-01 (P_tisAB+, b). When cells grow to exponential phase, they were exposed to 0 0.1 1 6 10 mg/L CIP in LB to induce the expression of P_tisAB. 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.

Next, we narrowed the range of CIP concentration and found the linear relationship between green fluorescence intensity and CIP concentration(Fig. 3). In this experiment, we chose wild-typeⅡ as the control (with an arabinose-inducible promoter), since arabinose-inducible promoter won’t be induced by CIP. No similar phenomenon was observed for the control group.

Fig. 3 Fold of Fluorescence Intensity per OD for E.coli DH5α carrying piGEM2019-01 (P_tisAB+) and control (P_tisAB-). When cells grow to exponential phase,they were exposed to 0 0.2 0.4 0.6 1.0 mg/L CIP in LB to induce the expression of P_tisAB. Fold means GFP unit fluorescence after 2h of exposure to CIP normalized to initial unit fluorescence. Unit fluorescence is fluorescence intensity per OD. 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 P_tisAB responds differently to CIP at different concentrations, and comparison shows that 1 mg/L is the most appropriate response concentration for P_tisAB.

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]Dörr, T., Vulić, M., & Lewis, K. (2010). Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli. PLoS biology, 8(2), e1000317.

Sequence and Features