Difference between revisions of "Part:BBa K3020000"

Revision as of 02:35, 29 September 2023

Green fluorescent protein reporter gene under oxidative damage-sensitive initiation

The SOS response is an inducing response when DNA replication is blocked or damaged. In E. coli, this reaction is regulated by the recA-lexA system, which does not function under normal physiological conditions. When DNA replication is blocked or damaged to produce an exposed single strand, the protease function of one of the RecA functions is immediately activated. The repressor protein LexA spontaneously degraded and sheds from the promoter of the SOS gene, thereby promoting the expression of genes involved in the SOS response (such as uvrA, uvrB, uvrC, uvrD, ssb, recA, and recN). Thereby, the functions of excision repair, post-replication repair and strand break repair related to these genes are generated, and a series of gene level and cell level responses are exerted.The expression levels of these SOS genes range from several times to tens of times when the SOS reaction does not occur. After the elimination of the inducing factor (such as a large number of DNA single strands), the protease activity of RecA disappears, and the amount of LexA protein is significantly increased, and the repression is re-acted. When the SOS reaction occurs, it can cause an increase in damage repair function in a short period of time. Since the expression of the SOS gene is closely related to the viability of its promoter, the signal of the reporter gene constructed under the gene promoter can indicate the activity of the promoter, and the signal size of the reporter factor and the concentration of the DNA damage reagent usually have a certain dose-effect relationship. Therefore, the SOS Promoter + Reporter System can be used as an indicator of the ability of a compound to detect DNA damage.

We successfully constructed SOS response induction by means of molecular cloning and synthetic biology.The plasmid consisting of the RecA promoter and the optimized eGFP fluorescent protein, the plasmid was transferred into the E. coli BL21 (DE3) expression vector, and its functionality was verified, which responded well to the DNA damaging agent and enhanced fluorescence. The protein can be correctly expressed and the plasmid can be transformed into different E. coli strains.

Target fragment electropherogram,The target segment is approximately 1100bp

Nalidixic acid is one of the antibacterial synthetic compounds, has a broad antibacterial spectrum which is particularly effective against Gram-negative bacteria, and is low in toxicity to animals and humans. It is known that bacteria have specificity for inhibiting DNA synthesis.Therefore, it can be used as an exogenous DNA damage reagent for the testing of genetic lines. The plasmid containing this line was transformed into competent cell BL21(DE3) and expanded to OD=0.2-0.4 .Inducing bacterial solution with 0, 10uM Nalidixic acid sodium salt for 2h and observed under a fluorescence microscope, It can be seen that the cells showed a very pronounced fibrosis when induced by 10 μmol/L NA for 2 hours, indicating that the cells stopped cell division. Since cell filamentation is one of the important features of the SOS reaction, it can be speculated that the strain cells have a significant SOS response at a lower concentration of NA treatment. It also demonstrates that the recA promoter successfully responds to DNA damage and controls the expression of the egfp gene in the SOS reaction, producing an enhanced green fluorescent protein that emits bright green fluorescence.

The plasmid responded to the sodium nalidixate reagent in the BL21 (DE3) strain.40× microscope under laser confocal fluorescence microscope, the strain grows to log phase,0 (A), 10 μM (B) NA was induced at 37 ° C for 2 hours. C and D are the corresponding bright field photos.

Relevant experimental results placed on the ‘experience’ page.

Contribution By PuiChing-Macau 2023

Sequence and Features