Difference between revisions of "Part:BBa K4691000"

Latest revision as of 09:35, 12 October 2023

recA-HRP-eGFP

SOS Response is a stress response of the organism itself, when bacterial DNA is damaged to a certain extent, it will repair the activity of related genes by activating the SOS response.In the genome of E. coli, many genes are found to participate in the damage repair process of SOS reaction, under normal circumstances, DNA damage repair gene activity is inhibited by LexA inhibitory protein, when SOS reaction occurs, RecA protein activity is activated, triggering the self-cleavage of LexA protein, and then initiating the transcriptional activity of downstream genes, expressing fluorescent proteins, observing bacterial growth to qualitatively evaluate biological damage effects, and using fluorescence signals to quantitatively evaluate DNA damage.

HrpRS and PhrpL are derived from the ultrasensitive pathogenic gene regulatory network (HRP) in the gram-negative bacterium Pseudomonas lilac, which enables high-gain transcription amplification.Specifically, the combination of HrpR protein and HrpS protein forms an ultrasensitive complex that binds to the upstream active sequence of the dependent σ54 factor hrpL promoter PhrpL, and uses the energy generated by ATP hydrolysis to transform the inactive σ54-RNAP-hrpL transcription complex into an active complex, thereby initiating the expression of downstream proteins and achieving high-gain signal output.

We combined the recA promoter and the HRP amplifier to construct the recA-HRP-eGFP gene circuit in anticipation of a more sensitive DNA damage sensor

Construct the plasmid

We obtained the recA-HRP-eGFP gene circuit by PCR and linked it to pUC19 vector by enzymatic ligation reaction.

Sensitivity test

Then we transferred this plasmid into BL21 competent cells.We applied 100μL on LB plate which contains 100 μg/mL of ampicillin resistance, cultured it upside down at 37℃ overnight. We selected the monoclonal colony, and expanded the cultures.After that we sent it to jinweizhi (Suzhou) biochemisty co., ltd for sequencing, and stored the remaining bacteria at -20℃ for reserve. （σ is the standard deviation of twenty blank group measurements，S is the slope of the linear equation） The HRP 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 h with gradient settings: 0μM, 0.3 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM. SFU was calculated to obtain the NA induction curve of HRP type bacteria.

From the curve, the linear interval for HRP bacteria expressing under NA induction is about 1~5μM, and the concentration gradient was refined in the linear interval and characterized again.

The overnight culture of HRP and wild-type bacteria 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 LOD of the HRP type sensor for NA is 0.01989μM, which is 1.8 times higher than the detection limit of 0.03580μM of the wild type sensor

Characterization Experiment with Different Damaging Agents

We validated the recognition range and discrimination ability of the sensor using different reagents.

Hydrogen peroxide, commonly used for sterilization, can cause DNA damage in bacteria. Acetone, a low-toxicity chemical, can precipitate proteins. KANA, a commonly used antibiotic in laboratories, can inhibit bacterial protein synthesis.

The HRP strain was induced for 2 h under different concentrations of NA, H₂O₂, acetone, and KANA.

The experimental results show that the strain exhibits a good response to DNA-damaging agents such as NA and H₂O₂. DNA damage caused by H₂O₂ concentrations of 100 μM, 500 μM, and 1000 μM corresponds to DNA damage caused by NA concentrations of 2 μM, 3 μM, and 4 μM, respectively. On the other hand, non-damaging agents such as acetone and KANA do not elicit a response from the strain. These results demonstrate that the constructed sensor can respond to different DNA-damaging agents and successfully differentiate between damaging and non-damaging agents.

Testing with Different Types of Damage

In addition to testing chemical agents, we were curious to see if the sensor's detection capabilities could extend to include electromagnetic radiation with thermal effects, as well as the field of ionizing radiation with potential for development. Ultraviolet (UV) lamps are commonly used for sterilization in biological laboratories. UV radiation can directly affect the DNA of biological cells, causing damage and leading to bacterial death. We first selected UV lamps to induce bacterial DNA damage and tested the sensor's response to radiation.

The overnight cultured HRP strain and wild-type strain were transferred to 30 mL fresh culture medium. Then, 30 μL of ampicillin was added, and the cultures were grown to the logarithmic growth phase. The strains were exposed to UV light for 0 minutes, 1 minute, 2 minutes, 3 minutes, 4 minutes, and 5 minutes. Subsequently, they were placed in a 37℃ shaker at 180 rpm, and the OD value and fluorescence intensity were measured every 20 minutes.

The OD values and fluorescence intensities of the two strains were taken after 1 min and 5 min of exposure to UV light. The RFU curves were calculated based on these measurements.

The experimental results indicate that the sensor can detect DNA damage caused by UV radiation. Furthermore, the HRP amplifier can enhance gene expression in this scenario as well, with amplification factors of up to 3-fold and 1.7-fold observed under 1 minute and 5 minutes of exposure, respectively.

Sequence and Features