Difference between revisions of "Part:BBa K629001"

Revision as of 15:06, 8 October 2023

recAp, could be started with exposure to irradiation, UV, nalidixic acid

Background

The recA gene of Escherichia coli is involved in genetic recombination, in postreplication repair, and in a number of other cellular functions such as mutagenesis, phage induction, and cell division. RecA protein is related to SOS system-- DNA repair, which is activated by recA promoter. The active level of recA promoter is fluctuates from low basal levels to very high levels after treatments that damage DNA or stall DNA replication. In our project, recA promoter is used to detect single-chain DNA which result from ionizing radiation, x-ray, ultraviolet radiation and some kinds of drug. According to the charater, ionizing radiation can activate recA promoter and raise the level of gene expression which under recA promoter's regulation.

Two exchangable ways to start recAp

Because of the special pathway in start of recAp, which is small pieces of break DNA can lead to the SOS system, there has been several ways to start this promoter from research and our try. In this case, the range of usage of this promoter could be spread.

1. UV light This way is based on the basic theroy of start of recAp, which means that when UV light damage DNA into some small pieces, recAp would be started.

What a pity in this method is that the parameters cannot be determined for the unstable data.

2. Nalidixic Acid

Nalidixic acid selectively and reversibly blocks DNA replication in susceptible bacteria. Nalidixic acid and related antibiotics inhibit a subunit of DNA gyrase and induce formation of relaxation complex analogue. It also inhibits the nicking dosing activity on the subunit of DNA gyrase that releases the positive binding stress on the supercoiled DNA. Nalidixic acid is effective against both gram-positive and gram-negative bacteria. In lower concentrations, it acts in a bacteriostatic manner; that is, it inhibits growth and reproduction. In higher concentrations, it is bactericidal, meaning that it kills bacteria instead of merely inhibiting their growth.

All in all, nalidixic acid inhibits DNA gyrase, blocking DNA replication and leading to degradation of DNA. As a result, it could be an optimal alternative for nuclear radiation. As a result, it has ability to start recAp.

In our several tests of this method, the concentration of NAL should between 5~25 ug/mL.

Contribution

iGEM2019[http://2019.igem.org/Team:BIT# BIT]

In 2019, IGEM_BIT team modified this part. We optimized the sequence of the recA promoter that bind to the sequence of lexA repressor protein in BBa_K629001(as shown in Figure 1). This allows the lexA protein to have a higher affinity for the recA promoter, and a stronger inhibition of downstream genes . Based on our SOS promoter + reporting factor system, the bottom noise is lower without damage, and the detection range is expanded. Since the modified RecA is located behind the ribosome binding site, even if the ribosome has been bound, it still inhibits its transcription, so that the degree of inhibition of the downstream gene is increased/ and the degree of expression of background fluorescence is lowered under the condition that the strain does not have SOS reaction. We used H2O2 as an inducer to induce strains, and the results are shown in Figure 2. The average error was taken three times for each experiment.

Figure 1:the comparison between BBa_K629001 and BBa_K3020001 transformation sequence

Figure 2: Induction factor comparison before and after recA promoter modification A is Response of strains to H2O2 before and after transformation，B is relative fluorescence intensity without inducer

Compared with the recA sequence before modification, the modified strain had an increase in the H2O2 inducing factor value of 1 mM and above. Figure 2B shows the relative fluorescence intensity of the bacteria when no exogenous damaging agent was added, that is, the degree of background fluorescence expression. Since the lexA repressor protein has stronger affinity with the promoter sequence and increased the inhibition of downstream genes, the degree of autofluorescence expression of the strain is reduced. The modified strain in this experiment successfully reduced the expression of the background and improved the sensitivity and the detection range.

Reference

[1] S. Nuyts et al., Radiation Research. 155, 716 (2001).

[2] S. I.Feinsteinl et al., Nucleic Acids Research. 11, 2927 (1983).

iGEM2023PuiChing-Macau

Hoping to validate previous iGEM team’s result and investigate the impact of the genotoxicity of carcinogens, we conducted dosage dependent tests on the top10 E.coli bacteria. RecA(K3), used with eGFP (composite part BBa_K302000) as bioreporter to detect DNA damaging agents such as UV, H2O2 and nalidixic acid, is the promoter derived from BBa_K3020001 by team BIT 2019. This promoter is an optimized version of BBa_K629001 (K6), which is a design of team SYSU 2011 aimed to drive the motor system under a radioactive environment.

We first treated the bacteria and transferred them into 96-well plates to record the fluorescence and optical density (absorbance at 600nm). However, as we discovered that this method might not be accurate enough, we chose to stabilize them on the microscope slides and took photos of the E.coli. After we processed these images, we calculated the mean value of any completely visible bacteria and used the data points from three independent experiments to plot the following box charts.

As can be shown in the following figures, the RecA(K6)-B0034-eGFP design does not illustrate the dosage dependence.

We then use Fluorescence over OD600 (FL over OD) to compare the EGFP signal in different groups. According to BIT 2019 (https://2019.igem.org/Team:BIT/Bio), SFU is used to compare fluorescence. (Specific fluorescence units SFU=RFU/OD600) After we compared the data of three independent experiments, we can observe that in both UV and H2O2, the SFU is directly proportional to the intensity/concentration of the carcinogen. In addition, the performance of K3 is better than K6, showing that the optimized promoter K3 reduced the background noise considerably.

Sequence and Features