Difference between revisions of "Part:BBa K629001"

Revision as of 05:53, 21 October 2019

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

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.

[File:BIT 2019 recA 1.png |460px]]

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).

Sequence and Features