Difference between revisions of "Part:BBa K4283013"
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| − | < | + | <h2>SulAp2.0 > double LexA Binding Unit RadiatioN Exercisable Regulator(dLexburner)</h2> |
| − | Here we report an improved version of sulAp UV-inducible promoter, the double LexA Binding Unit RadiatioN Exercisable Regulator (dLexburner | + | 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). This part like sulAp, could be used in all prokaryotic organisms with LexA-RecA stress response SOS systems (i.e. E. coli) |
| − | < | + | <h3>Mechanism of sulAp</h3> |
<b>Figure 1 </b> | <b>Figure 1 </b> | ||
| − | [[File:Fig 1.png|600px|thumb|center|illustrative diagram of the LexA-RecA | + | [[File:Fig 1.png|600px|thumb|center|illustrative diagram of the LexA-RecA stress-inducible expression system in E. coli.]] |
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| + | <p>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.</p> | ||
<b>Figure 2 </b> | <b>Figure 2 </b> | ||
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[[File:Fig 2.png|600px|thumb|center|Structure of dLexburner, consists of two LBS, using the same promoter element as sulAp.]] | [[File:Fig 2.png|600px|thumb|center|Structure of dLexburner, consists of two LBS, using the same promoter element as sulAp.]] | ||
| − | < | + | <h3>Characterization</h3> |
<p>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.</p> | <p>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.</p> | ||
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<p>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.</p> | <p>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.</p> | ||
| − | < | + | <p>We have yet assayed the effect of changing homogeneity of the host LexA gene on the sensitivity of this component. However, LexA and RecA are both highly conserved in E. coli, thus this component shows promising capability of applying presicer optogenetic controls with less leaky expression for projects interested in future.</p> |
===Usage and Biology=== | ===Usage and Biology=== | ||
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Latest revision as of 10:24, 13 October 2022
dLexburner SulAp
SulAp2.0 > double LexA Binding Unit RadiatioN Exercisable Regulator(dLexburner)
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). This part like sulAp, could be used in all prokaryotic organisms with LexA-RecA stress response SOS systems (i.e. E. coli)
Mechanism of sulAp
Figure 1
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
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.
We have yet assayed the effect of changing homogeneity of the host LexA gene on the sensitivity of this component. However, LexA and RecA are both highly conserved in E. coli, thus this component shows promising capability of applying presicer optogenetic controls with less leaky expression for projects interested in future.