Difference between revisions of "Part:BBa K2705010"
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<partinfo>BBa_K2705010 short</partinfo> | <partinfo>BBa_K2705010 short</partinfo> | ||
| − | P<sub><i>liaG&43- | + | P<sub><i>liaG&43-1</i></sub> is a promoter which is an artificial combination of the σ<sup>A</sup> recognition site of P<sub><i>liaG</i></sub> and the σ<sup>B</sup> recognition site of P<sub><i>43</i></sub> with modification. |
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===The original part P<sub><i>liaG</i></sub> (BBa_K823000)=== | ===The original part P<sub><i>liaG</i></sub> (BBa_K823000)=== | ||
| − | P<sub><i>liaG</i></sub> ( | + | P<sub><i>liaG</i></sub> ([https://parts.igem.org/Part:BBa_K2705000 BBa_K2705000]) is a weak, constitutive promoter from <i>B. subtilis</i>. It’s responsible for the transcription of the last four genes of the <i>liaIHGFSR</i> locus and therefore for the production of the components of the LiaRS system, which is important for the detection of cell wall antibiotics (Jordan et al., 2006). P<sub><i>liaG</i></sub> was evaluated with the lux operon as well as the <i>lacZ</i> as reporter. For more details, visit the Data page of the LMU-Munich Team 2012 or get an overview of the whole project Beadzillus.( http://2012.igem.org/Team:LMU-Munich) |
===Design=== | ===Design=== | ||
| − | We intended to improved the part P<sub><i>liaG</i></sub> ( | + | We intended to improved the part P<sub><i>liaG</i></sub> ([https://parts.igem.org/Part:BBa_K2705000 BBa_K2705000]). Inspired by the relatively strong constitutive promoter P<sub>43</sub>([https://parts.igem.org/Part:BBa_K143013 BBa_K143013]), we chose to mimic its structure, which can be recognized by both sigma factor 55 (the major sigma factor A) and sigma factor 37 (the lag phase sigma factor B). As the promoter P<sub><i>liaG</i></sub> is a constitutive <i>Bacillus subtilis</i> σ<sup>A</sup> promoter, we replace some part of the promoter P<sub><i>liaG</i></sub> sequence with the σ<sup>B</sup> recognition site at the same relative position which the σ<sup>B</sup> recognition site to the σ<sup>A</sup> recognition site of the promoter P<sub>43</sub> to create the new promoter P<sub><i>liaG&43</i></sub>. On the basis of this, we made some adjustments to the sequence of the -35 region of P<sub><i>liaG</i></sub> (from CGTATC to TTGACA) to create another new promoter P <sub><i>liaG&43-2</i></sub> (BBa_K2705010). |
===Luminescence measurements=== | ===Luminescence measurements=== | ||
| − | The constitutive promoters P <sub><i>liaG&43-2</i></sub> (BBa_K2705010) was evaluated in the reporter vector pHT01-P<sub><i>x</i></sub>-GFP. The promoter activity results in gene expression and production of the green fluorescence protein(GFP). The fluorescence intensity(FI) was measured by the Multimode Plate Reader <i>Enspire</i>(PerkinElmer). | + | The constitutive promoters P <sub><i>liaG&43-2</i></sub> ([https://parts.igem.org/Part:BBa_K2705010 BBa_K2705010]) was evaluated in the reporter vector pHT01-P<sub><i>x</i></sub>-GFP. The promoter activity results in gene expression and production of the green fluorescence protein(GFP). The fluorescence intensity(FI) was measured by the Multimode Plate Reader <i>Enspire</i>(PerkinElmer). |
All clones showed a normal growth behavior. As the σ<sup>B</sup> recognition site of P<sub>43</sub> we added is for the lag phase, we tested the FI at the very beginning (the lag phase, Fig.1) of its life. P <sub><i>liaG&43-2</i></sub> outperformed the original promoter P <sub><i>liaG</i></sub> at the lag phase, whose activity is slightly higher than that of P <sub><i>liaG</i></sub>. | All clones showed a normal growth behavior. As the σ<sup>B</sup> recognition site of P<sub>43</sub> we added is for the lag phase, we tested the FI at the very beginning (the lag phase, Fig.1) of its life. P <sub><i>liaG&43-2</i></sub> outperformed the original promoter P <sub><i>liaG</i></sub> at the lag phase, whose activity is slightly higher than that of P <sub><i>liaG</i></sub>. | ||
[[File: Fig. 1 new Luminescence measurement of the constitutive Bacillus sp. promoters P liaG&43-2 and PliaG in the reporter vector pHT01-Px-GFP.png|600px|center|thumb|''' Fig. 1 Luminescence measurement of the constitutive <i>Bacillus</i> sp. promoters P <sub><i>liaG&43-2</i></sub> and P <sub><i>liaG</i></sub> in the reporter vector pHT01-P<sub><i>x</i></sub>-GFP.''' The strains were cultured in LB medium with 5 µg/mL chloramphenicol in dark for 2 hours.Fluorescence intensity of GFP was measured, which was normalized against OD<sub>600</sub>. Data indicate mean values ± standard deviations from three independent experiments performed in triplicates.]] | [[File: Fig. 1 new Luminescence measurement of the constitutive Bacillus sp. promoters P liaG&43-2 and PliaG in the reporter vector pHT01-Px-GFP.png|600px|center|thumb|''' Fig. 1 Luminescence measurement of the constitutive <i>Bacillus</i> sp. promoters P <sub><i>liaG&43-2</i></sub> and P <sub><i>liaG</i></sub> in the reporter vector pHT01-P<sub><i>x</i></sub>-GFP.''' The strains were cultured in LB medium with 5 µg/mL chloramphenicol in dark for 2 hours.Fluorescence intensity of GFP was measured, which was normalized against OD<sub>600</sub>. Data indicate mean values ± standard deviations from three independent experiments performed in triplicates.]] | ||
Latest revision as of 15:38, 17 October 2018
PliaG&43-2
PliaG&43-1 is a promoter which is an artificial combination of the σA recognition site of PliaG and the σB recognition site of P43 with modification.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
The original part PliaG (BBa_K823000)
PliaG (BBa_K2705000) is a weak, constitutive promoter from B. subtilis. It’s responsible for the transcription of the last four genes of the liaIHGFSR locus and therefore for the production of the components of the LiaRS system, which is important for the detection of cell wall antibiotics (Jordan et al., 2006). PliaG was evaluated with the lux operon as well as the lacZ as reporter. For more details, visit the Data page of the LMU-Munich Team 2012 or get an overview of the whole project Beadzillus.( http://2012.igem.org/Team:LMU-Munich)
Design
We intended to improved the part PliaG (BBa_K2705000). Inspired by the relatively strong constitutive promoter P43(BBa_K143013), we chose to mimic its structure, which can be recognized by both sigma factor 55 (the major sigma factor A) and sigma factor 37 (the lag phase sigma factor B). As the promoter PliaG is a constitutive Bacillus subtilis σA promoter, we replace some part of the promoter PliaG sequence with the σB recognition site at the same relative position which the σB recognition site to the σA recognition site of the promoter P43 to create the new promoter PliaG&43. On the basis of this, we made some adjustments to the sequence of the -35 region of PliaG (from CGTATC to TTGACA) to create another new promoter P liaG&43-2 (BBa_K2705010).
Luminescence measurements
The constitutive promoters P liaG&43-2 (BBa_K2705010) was evaluated in the reporter vector pHT01-Px-GFP. The promoter activity results in gene expression and production of the green fluorescence protein(GFP). The fluorescence intensity(FI) was measured by the Multimode Plate Reader Enspire(PerkinElmer). All clones showed a normal growth behavior. As the σB recognition site of P43 we added is for the lag phase, we tested the FI at the very beginning (the lag phase, Fig.1) of its life. P liaG&43-2 outperformed the original promoter P liaG at the lag phase, whose activity is slightly higher than that of P liaG.
