Latest revision as of 10:14, 12 October 2023

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 2019iGEM_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，figure B is the relative fluorescence intensity value without DNA damaging agents

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.

Contribution

By PuiChing-Macau 2023

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. This part RecA(K3) is used with eGFP (BBa_K3020002) as bioreporter (RecA(K3)-B0034-eGFP) to test the performance of K3 and compare it with K6 (BBa_K629001) promoter. We used it to detect DNA damaging agents such as UV, H2O2 and nalidixic acid.

Imaging Analyze Result

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(K3)-B0034-eGFP design illustrates the dosage dependence.

SFU (FL over OD) graphs

We also used 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.

RBS test

We also tried different RBS (strong and B0032) to replace the B0034 (in BBa_K4814015, the one we designed to test the performance of K3 promoter). The result indicated that RecA(K3)-eGFP with B0032 RBS has a stronger SFU (FL over OD600) than the original B0034 we chose. Please refer to BBa_K4814002 (B0032) and BBa_K4814013 (Strong RBS) for details.

RBS test of UVB and H2O2 FL over OD600 of RecA(K3)-B0034-eGFP, RecA(K3)-B0032-eGFP, and RecA(K3)-Strong-eGFP.

RBS test of Nalidixic acid and Aspartame FL over OD600 of RecA(K3)-B0034-eGFP, RecA(K3)-B0032-eGFP, and RecA(K3)-Strong-eGFP.

Sequence and Features