Part:BBa_K3020000:Experience

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Typical DNA damage agents include nalidixic acid (NA) (Sigma), formaldehyde (formaldehyde, CH2O) (Sigma) and hydrogen peroxide(H2O2).The following non-DNA damage reagent was used as a negative control:kanamycin sulfate，KAN）（Amresco）,acetone,phenol. The reagent concentration and gradient were calculated and added to the 40 ml bacterial solution (LB) system according to the following induction volume.

DNA damage agent concentration gradient and induced addition

non-DNA damage agent concentration gradient and induced addition

The BL21 strain introduced with Part:BBa_K3020000 was cultured overnight in LB medium (AMP resistance) at 37°C, and inoculated into fresh LB liquid medium (AMP resistance) at 1:50 inoculation, and shake cultured at 37 ° C for 3 hours. The OD600 is 0.2-0.4. Add DNA damaging agents and non-injury agents with different concentration gradients. The concentration and dosage are shown in the above figure. Induction at 37°C 130 r / min for 2-3 h. Standard error was taken three times for each set of experiments.

date analysis

OD600 represents the absorbance or optical density of a sample measured at a wavelength of 600 nm, which is a common method for estimating the concentration of bacteria or other cells in a liquid. The measured concentration can indicate the stage of culturing the cell population, ie whether it is in a lag phase, an exponential phase or a stationary phase.

Relative optical density ratio ROD=ODx/OD0

The formula is the relative optical density ratio calculation formula, OD x is the OD 600 value of the experimental group, OD0 is the OD 600 value of the control group without adding the damaging agent at the same time, and the ROD reflects the toxicity to the bacteria exposed to known or unknown compounds.

specific fluorescence units SFU=RFU/OD 600

The formula is the specific fluorescence intensity, where RFU is the relative fluorescence intensity unit measured by the microplate reader, OD 600 is the OD value of the same sample, representing the fluorescence intensity per unit OD, and RFU reflects the fluorescent protein expression intensity of the cells. This unit avoids deviations in the calculation of fluorescence intensity due to differences in the number of bacteria between different samples.

induction factor Fi=SFUx/SFU0

Where SFUx is a sample treated with a toxic compound and SFU0 is a control sample at the same time point. In this experiment, the inducing factor Fi value was used to evaluate the ability of the compound to damage the reagent. It can be seen from the literature that when the induction time is 2 h, the Fi factor value of any compound at any concentration reaches 2 or higher. This compound was identified as a DNA damaging agent.

results and discussion

1.Response of strains to DNA damage reagents

Nalidixic acid inhibits DNA replication and topoisomerase II, which is commonly used to kill bacteria;formaldehyde crosslinks DNA strands, DNA single strand breaks, usually as a formalin solution; H2O2 usually Used for disinfection and bleaching of teeth, causing DNA oxidative damage. In order to detect the response of the experimental strain to the induction of toxic compounds, the fluorescence spectrum at 516 nm was scanned at room temperature in a black 96-well plate after induction. The relationship between the induction factor Fi and the optical density ratio ROD and the different induced concentrations were plotted. According to the definition in the literature, this compound was identified as a DNA damaging agent when the F i value was greater than or equal to 2 at any concentration after 2 h of induction. It can be seen from the figure below that different DNA damage agents induce experimental strains under different concentration gradients, and the unit fluorescence intensity expressed by the strain increases with increasing concentration. However, under the induction of 0.01 M H2O2, excessive cytotoxicity resulted in excessive cell death, resulting in a decrease in fluorescence intensity.

1.Different concentrations of damaging agents induce fluorescence intensity

The experimental strains showed a significant dose-fluorescence effect relationship in the injured compounds under these different concentration gradients (see the left panel below), indicating that this recA-eGFP line sensor has a good response to different damaging agents. In the left panel, the inducing factor value of the experimental strain gradually increased with the increase of the concentration of the DNA damage reagent, and in the case of H2O2 induction, the inducing factor of the strain increased first and then decreased, indicating that it was induced at a higher concentration，the toxicity of the cells is greater than the DNA damage to the strain.

2.The logarithmic phase of the bacterial strain was induced at different concentrations of different damaging agents for 2 h.The reference line in the figure is 2.

3.The change in the optical density ratio of the strain under the induction of the damaging agent.

2.Response of strains to non-DNA damage reagents

Acetone is a common organic solvent that has an anesthetic effect on the human central nervous system. Exposure of cells to acetone causes protein precipitation. Kanamycin sulfate is a commonly used antibiotic in the laboratory. It is commonly used for the screening of resistant strains, which can inhibit the production of proteins and cause misinterpretation of mRNA codes by binding to 30S ribosomes. Phenol is highly corrosive to human skin and mucous membranes and can denature local proteins. In this section, the effect of concentration on the induction factor Fi is obtained by the induction of different non-DNA damage agents by experimental strains, as shown in the figure.

4.Response of strains to non-DNA damage reagents

From the comparison of Fig. 3 and Fig. 4B, the concentration of the two types of reagents increases, and the ratio of the optical density of the bacterial liquid decreases, indicating that both types of reagents have strong cytotoxicity to the experimental strain regardless of whether or not DNA damage is caused. When the strain was treated with a non-DNA damaging agent, the induction factor was less than 2 even when induced at a high concentration, indicating that several reagents selected did not have the ability to damage DNA, and none of them could cause SOS reaction. In addition, it has successfully demonstrated that the experimental strain can distinguish between DNA damage reagents and non-damage reagents, and can detect the DNA damage ability as well as the toxicity of the reagents and more unknown compounds to the cells.

LOD measured by BIT 2023

In order to intuitively obtain the detection performance of the biosensor, we use LOD (limit of detection) as an indicator to supplement the detection limit data of the BBa_K3020000.

The bacteria cultured overnight was transferred to 30 ml of fresh LB liquid medium, cultured to the logarithmic growth phase after adding 30μL ampicillin. Add different concentrations of DNA damaging agent NA and culture for 2.5 h with gradient settings: 1 μM, 2 μM, 3 μM, 4 μM, 5 μM. Fit the output signal to a linear equation. According to the formula LOD=3σ/S （σ is the standard deviation of twenty blank group measurements，S is the slope of the linear equation）, the lLOD of the biosensor for NA is 0.03580μM.