Latest revision as of 10:25, 13 October 2022

sulA promoter

Microbes including Escherichia coli are known to respond to various DNA-injuring stress (ionizing radiation, ultraviolet radiation, peroxides etc...), altering their gene expression patterns. This response, known as the "SOS response", is induced by a regulatory protein called RecA when it binds to single-strand DNA. The DNA-RecA complex promotes the degradation of LexA, a common repressor of SOS genes.

SulA is responsible for stress-induced halt of cell division. The promoter of sulA, sulAp, is induced by various stress factors, including ultraviolet irradiation.

Application

We utilized this property (induced-expression by UV irradiation) to design a "UV switch". This makes it possible to "switch on" a genetic circuit using UV. As a first step for this, we characterized the UV-induction of sulAp.

The UV-induced expression level of BBa_K518010 was evaluated using BBa_K518013. As a measurement tool, our dual luciferase assay kit was employed. For detailed infomation, see BBa_K518002.

<Figure: UV-induced expression levels of sulAp. The expression levels from sulAp were evaluated before and after UV induction. We evaluated it using both recA(-) (JM109) and recA(+) (BL21) strain. RecA is known to be necessary for releasing sulAp from repression. We successfully demonstrated a significant alteration of expression in recA(+) strain only after UV irradiation. The expression level of BBa_J23119, a constitutive E. coli promoter which is often used as a comparison, was simultaneously presented. Data is expressed as mean ± S.D.. Data is obtained from the average of three independent experiments.>

Team: 2020 SZ-SHD: Investigated the UV-induction time to the expression level of SulAp:

In our project, experiments have been carried out to measure the expression level of eGFP gene behind SulAp promoter in BL21, with different exposure time under UV induction. Activity of SulAp with and without UV induction has been investigated with the aid of the gene for green fluorescence protein (eGFP). We ligated the pSulA-eGFP fragment onto vector pSB1C3, which then transformed into the BL21 (E. coli). The increase in fluorescence can be observed over 8 hours after induction. Where there were 3-4 times increments in fluorescence than normal, as displayed in the diagram above.

Figure: the result of eGFP expression after irradiated under UVC, unit: fluorescence per OD. eGFP- UV+: non-recombinant BL21 irradiated under UVC (without pSS1); eGFP- UV-: non-recombined BL21 without UV exposure. 0min: recombined BL21, no UV exposure; 0.5min: recombined BL21 irradiated under UV for 30 seconds; 1min: irradiated under UV for 1 min; 2min: irradiated under UV for 2 min.

More information:

more information on this part
Sequence and Features

SZ-SHD2022: Characterization of UV promoter sulAp

Activity of SulAp with and without UV induction has been investigated with the aid of the gene for green fluorescence protein (eGFP). We ligated the pSulA-eGFP fragment onto vector pSB1C3, which then transformed into the top10 (E. coli).The increase inFluorescence per OD600 will be measure through a plate reader.

Figure: The result of eGFP expression after irradiated by 15mW/cm2 UVC for 1min Blank: Ecoli Top10 strain without vector UV-: pSB1C3-SulAp-eGFP(top10) without UV radiation UV+: pSB1C3-SulAp-eGFP(top10) irradiated under UV.

Based on the data, we improved this part by adding one more LexA binding site, new part: BBa_K4283013

SZ-SHD 2022: Improvement of this part

SulAp2.0 > double LexA Binding Unit RadiatioN Exercisable Regulator(dLexburner SulAp)

Here we report an improved version of sulAp UV-inducible promoter, the double LexA Binding Unit RadiatioN Exercisable Regulator (dLexburner,). SulAp has been used for regulatory purposes and was initially designed and submitted by team UT-Tokyo in 2011 (iGEM11_UT-Tokyo, Part:BBa_K518010).

Mechanism of sulAp

Figure 1

illustrative diagram of the LexA-RecA co-operative stress-inducible expression system in E. coli. LexA as a expression repressor is constitutively produced by E coli from a conserved genomic area, which binds to specific LexA binding sequences found in a few promoters. External stress will higher the activity of RecA in E. coli to degrade LexA and de-repress the gene expression downstream.

Figure 2

Structure of dLexburner, consists of two LBS, using the same promoter element as sulAp.

Characterization

We have evaluated the promoter activity by placing an eGFP gene downstream of dLexburner. The absorbance of green light (excitation 485nm, read at 520nm) was measured per OD600 of E. coli LB culture over hours after irradiated under 254nm UV light (15 mWcm(-2)). The relative intensity of fluorescence reflects the amount of GFP expressed and thus the activity of the promoter could be predicted.

Figure 3

A significant increment in fluorescence was observed for dLexburner in 2hrs after 1min UV exposure. In comparing with the sulAp, the basal expression level of dLexburner is only 0.75 times the initial value but very similar in hight in when plateaued. Therefore the approximate promoter activation is 5-6 times the basal level comparing to 2-3 times for sulAp.