Team:BIT/Demonstrate
Demonstrate
1 Pre-test
First we got the umuDC, dinD, dnaK by the PCR.
Then we constructed the corresponding gene lines and verified them.
Culture the sensor strain to the logarithmic phase, add the different concentrations of NA to the 40mL bacterial liquid of equal volume, and culture at 37℃ for 2h and 3h at 130r/min. NA can inhibit DNA replication and topoisomerase II, which is usually used to kill bacteria.
We got the data.
The experimental strains tell that DNA damaging agent NA can`t cause a significant drop in the protein expression efficiency of E. coli.
The experimental strains “umuDC+GFP” use more sensitive results tell us that DNA damaging agent NA can`t cause a significant drop in the protein expression efficiency of E. coli. But in a low concentration, the protein expression efficiency drops. We are looking for an answer.
So we can make sure our gene lines can play a role in the E.coli. And the the experimental strains give us good results.
We can see that the experimental strains showed a significant dose-fluorescence relationship in the damaged compounds under different concentration gradients. As shown in the figure, the dinD-GFP line sensor has a good response to different damage agents. But in the case of NA induction, the SFU of the strain first increased, then decreased and finally increased again. We think it is caused by the dinD promoter`s function cause the fact.
So we choose to use recA-eGFP line sensor to do the next work.
2 Construction and representation of gene circuits, sensors and bacterial strain
Enzyme digest the constructed gene line for verification. Theoretically, the length of recA+eGFP fragment is about 1100bp. The experimental results agree with the theory.
Send the clone positive plasmid to the company for sequencing. The sequencing primer is the universal primer of pUC19.
- The sequencing primer at 5’: M13F: TGTAAAACGACGGCCAGT;
- The sequencing primer at 3’: M13R: CAGGAAACAGCT ATGACC.
The sequencing results showed that the plasmid construction was successful. The genetic map is shown below.
Observe and photograph the NA-induced strains under the laser scanning confocal microscope. Compare figure A with figure B, we can see that after induced by 10μM naladixic acid for 2 hours, the cells are clearly filamentated, which shows that cells stop dividing. Because filialization is an important characteristic of SOS response, we can infer that the SOS response appears in the bacterial strain induced by 10μM naladixic acid. At the same time, the constructed SOS response-induced recA promoter and the gene circuit of eGFP fluorescence expressing protein successfully responded to the DNA damage agent, and the promoter successfully expressed the reporter protein in the reaction.
3 Response of the bacterial strain to DNA damage reagent
Culture the sensor strain to the logarithmic phase, add the different concentrations of NA, H2O2 and CH2O to the 40mL bacterial liquid of equal volume, and culture at 37℃ for 2h at 130r/min. NA can inhibit DNA replication and topoisomerase II, which is usually used to kill bacteria. Formaldehyde crosslinks DNA strands and breaks single DNA strands, usually as a preparation of formalin solution. H2O2 is commonly used to disinfect and bleach teeth and can cause oxidative damage to DNA.
To test the response of experimental strains to toxic compounds，put the inducted strain in the black 96-well plate at room temperature and scan the fluorescence spectra at 516nm and make the relationship curves between Fi and ROD and different inducible concentrations respectively. According to the literature definition in PROtocol, if Fi is bigger than or equal to 2 at any concentration after 2 hours induction, this compound can be identified as DNA damage agent.
It can be seen from the figure that under different concentration gradients, different DNA damage agent induced strains showed higher unit fluorescence intensity with the increase of concentration. However, under the induction of 0.01M H2O2, excessive toxicity led to excessive death of bacteria, resulting in a decrease in fluorescence intensity.
The experimental strains showed a significant dose-fluorescence relationship in the damaged compounds under different concentration gradients. As shown in the figure, the recA-eGFP line sensor has a good response to different damage agents. In figure 6 (A), as the test concentration of DNA damage reagent increased continuously, the induced factor of the experimental strain gradually increased. However, in the case of H2O2 induction, the induced factor of the strain first increased and then decreased, indicating that the toxicity of the cell was greater than the DNA damage of the strain under higher concentration induction.
4 Detection limit of the bacterial strain to DNA damage agent
As the figure 6 (A)，when induced by 1μM NA, Fi=1.59599±0.05016; when induced by 5μM NA, Fi=3.29409±0.067684; when induced by 10μM NA, Fi=4.76834±0.02262. Do the linear equation by three points (figure 7).
In formula (1), y is the induction factor and x is the concentration under the induction of NA. According to the definition of the minimum detection limit, when y = 2, the minimum detection limit of strains to NA is 1.95μM, and the response of the experimental strain to NA was significantly lower than strain sensors from the literature: 3.57μM. This experiment strains showed lower detection limit and higher sensitivity than the literature reported microbial sensor used for damage detection.Fi increased with the increase of NA concentration, showing a significant relationship between dose and fluorescence effect.
5 Response of bacterial strains to non-dna damage agents
Acetone, as a common organic solvent, has an anesthetic effect on the central nervous system of human beings. Exposure of cells to acetone causes the deposition of proteins. Kanamycin sulfate is a commonly used antibiotic in the laboratory. It is often used to screen for resistant strains, which can inhibit the production of protein and cause the misunderstanding of the mRNA code by binding to 30S ribosomes. Phenol is highly corrosive to human skin and mucous membranes and can denaturate local proteins. In this section, we obtain the effect relationship between the concentration and the induced factor through the induction of different non-dna damage agents by experimental strains, as shown in figure 7.
As shown in figure 6B and figure 8B, when the concentration of the two reagents increased, the OD of the bacterial solution decreased, indicating that both reagents had strong cytotoxicity to the experimental strains regardless of whether DNA damage was caused. However, when the strains were treated with non-dna damage agents, the inducing factors were all less than 2, even at a high concentration, indicating that the selected reagents did not have the ability to cause DNA damage and could not cause SOS response. In addition, it has also been successfully proved that the experimental strain can distinguish DNA damage reagent from non-damage reagent, and can detect the DNA damage ability as well as the toxicity of the reagent and more unknown compounds to cells.
Conclusion
- 1.We successfully cloned the SOS response-induced recA promoter to the upstream of eGFP fluorescent protein, and controlled the expression of eGFP fluorescence through the activation of recA promoter.
- 2.We used different DNA damage agents to induce the SOS response of the strain so as to establish the relationship between the concentration and dose of DNA damage agents and the degree of fluorescence expression
- 3.The sensor can successfully distinguish damage from non-damage agents and can effectively evaluate the toxicity of toxic agents to cells.
- 4.This sensor is intended to be tested on a microfluidic chip and then reflected to the user through a smart phone. See hardware section for details.