Part:BBa_K3020000

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 812
  
• 1000
  COMPATIBLE WITH RFC[1000]