Part:BBa_K4691000
recA-HRP-eGFP
SOS Response is a stress response of the organism itself, when bacterial DNA is damaged to a certain extent, it will repair the activity of related genes by activating the SOS response.In the genome of E. coli, many genes are found to participate in the damage repair process of SOS reaction, under normal circumstances, DNA damage repair gene activity is inhibited by LexA inhibitory protein, when SOS reaction occurs, RecA protein activity is activated, triggering the self-cleavage of LexA protein, and then initiating the transcriptional activity of downstream genes, expressing fluorescent proteins, observing bacterial growth to qualitatively evaluate biological damage effects, and using fluorescence signals to quantitatively evaluate DNA damage.
HrpRS and PhrpL are derived from the ultrasensitive pathogenic gene regulatory network (HRP) (HRP) in the gram-negative bacterium Pseudomonas lilac, which enables high-gain transcription amplification.Specifically, the combination of HrpR protein and HrpS protein forms an ultrasensitive complex that binds to the upstream active sequence of the dependent σ54 factor hrpL promoter PhrpL, and uses the energy generated by ATP hydrolysis to transform the inactive σ54-RNAP-hrpL transcription complex into an active complex, thereby initiating the expression of downstream proteins and achieving high-gain signal output.
We combined the recA promoter and the HRP amplifier to construct the recA-HRP-eGFP gene circuit in anticipation of a more sensitive DNA damage sensor
We obtained the recA-HRP-eGFP gene circuit by PCR and linked it to pUC19 vector by enzymatic ligation reaction.
Then we transferred this plasmid into BL21 competent cells.We applied 100μL on LB plate which contains 100 μg/mL of ampicillin resistance, cultured it upside down at 37℃ overnight.
We selected the monoclonal colony, and expanded the cultures.After that we sent it to jinweizhi (Suzhou) biochemisty co., ltd for sequencing, and stored the remaining bacteria at -20℃ for reserve.
The HRP bacteria cultured overnight was transferred to 30 ml of fresh LB liquid medium, cultured to the logarithmic growth phase after adding 30μL ampicillin. Add different concentrations of DNA damaging agent NA and culture for 2 h with gradient settings: 0μM, 0.3 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM. SFU was calculated to obtain the NA induction curve of HRP type bacteria.
From the curve, the linear interval for HRP bacteria expressing under NA induction is about 1~5μM, and the concentration gradient was refined in the linear interval and characterized again.
The overnight culture of HRP and wild-type bacteria was transferred to 30 ml of fresh LB liquid medium, cultured to the logarithmic growth phase after adding 30μL ampicillin. Add different concentrations of DNA damaging agent NA and culture for 2.5 h with gradient settings: 1 μM, 2 μM, 3 μM, 4 μM, 5 μM. Fit a linear equation. According to the formula LOD=3σ/S, the detection limit of the HRP type sensor for NA is 0.01989μM, which is 1.8 times higher than the detection limit of 0.03580μM of the wild type sensor
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2010
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1220
Illegal BsaI.rc site found at 2893
Illegal SapI.rc site found at 1853
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