Difference between revisions of "Part:BBa K3020001"

Revision as of 15:21, 20 October 2019

recA promoter-Respond to sos response and initiate expression of downstream repair proteins

This part is improved from iGEM2011_SYSU-China’s part BBB_K629001 by BIT in 2019, and it is one of the gold award criteria. More details can be found at https://igem.org/2019_Judging_Form?id=3020

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. More details can be found at BBa_K3020000。

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.

Sequence and Features