Difference between revisions of "Part:BBa K1696005"
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''BL21'' transferred with pET-28a(+)-P170-sfGFP and ''BL21'' transferred with pET-28a(+) were inoculated in LB medium with pH 5 and pH 7 respectively. we measured the Fluorescence intensity using fluorescence intensity detection microplate reader. The result is showed in Fig.3.The result showed that the fluorescence intensity of ''BL21'' with pET-28a(+)-P170-sfGFP t was much higher than control ''BL21''. In pH 5, the fluorescence intensity using prompter P170 is 123.693. while in pH7, the fluorescence intensity using P170 is 101.407.
 
''BL21'' transferred with pET-28a(+)-P170-sfGFP and ''BL21'' transferred with pET-28a(+) were inoculated in LB medium with pH 5 and pH 7 respectively. we measured the Fluorescence intensity using fluorescence intensity detection microplate reader. The result is showed in Fig.3.The result showed that the fluorescence intensity of ''BL21'' with pET-28a(+)-P170-sfGFP t was much higher than control ''BL21''. In pH 5, the fluorescence intensity using prompter P170 is 123.693. while in pH7, the fluorescence intensity using P170 is 101.407.
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[[File：P170 1.png|center|500px]]
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[[File:P170_1.png|center|500px]]

Revision as of 08:38, 20 October 2019

P170 Promoter

P170 is upregulated at low pH (6.0~6.5) during the transition to stationary phase. The minimal DNA region required for both promoter activity and pH regulation was mapped to a 51 bp fragment located 7 bp upstream of the transcriptional start site. This fragment lacked the consensus -35 promoter region, but it contained an ‘extended’ -10 promoter region[1]. Besides, a novel 14 bp regulatory DNA region centered at around -41.5 and composed of three tetra nucleotide sequences, boxes A, C and D. Boxes A and C contribute to P170 activity, whereas box D and the position of boxes ACD (renamed ACiD-box) are essential to P170 activity and acid response[2].


Reference

[1] Madsen S M, Arnau J, Vrang A, et al. Molecular characterization of the pH-inducible and growth phase-dependent promoter P170 of Lactococcus lactis.[J]. Molecular Microbiology, 1999, 32(1):75-87.

[2] Søren M. Madsen, Thomas Hindré, Jean-Paul Le Pennec, et al. Two acid-inducible promoters from Lactococcus lactis require the cis-acting ACiD-box and the transcription regulator RcfB[J]. Molecular Microbiology, 2005, 56(3):735-746.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


2019-SZPT-CHINA

First, we inserted P170-sfGFP the E. coli expression vector pET-28a(+). Fig.1 showed that the P170-sfGFP with size 1251bp was cloned to pET-28a(+) vector successfully.

P170.png

Then the recombinant vector pET-28a(+)-P170-sfGFP was transformed into E. coli BL21. The recombinant strain BL21 with pET-28a(+)-P170-sfGFP was selected and inoculated in LB medium with pH 5. BL21 with pET-28a(+) was used as control. After Incubate for 18 h at 37 ° C. The bacterium was placed under UV light to excite sfGFP. The results are shown in the Fig.2. The green sfGFP from BL21 transferred with pET-28a(+)-P170-sfGFP of sfGFP be seen obviously in pH 5.

BL21 transferred with pET-28a(+)-P170-sfGFP and BL21 transferred with pET-28a(+) were inoculated in LB medium with pH 5 and pH 7 respectively. we measured the Fluorescence intensity using fluorescence intensity detection microplate reader. The result is showed in Fig.3.The result showed that the fluorescence intensity of BL21 with pET-28a(+)-P170-sfGFP t was much higher than control BL21. In pH 5, the fluorescence intensity using prompter P170 is 123.693. while in pH7, the fluorescence intensity using P170 is 101.407.

P170 1.png