Part:BBa_K4202025

Pveg-1X leader RNA for Bacillus subtilis

A leader RNA is ligated to PsacB promoter and spoVG RBS downstream. Different sucrose concentrations can induce the expression of promoters. This device is planned to be used by ZJU-China 2022 for sucrose-controlled induction of expression of a large number of proteins in Bacillus subtilis.

Result

We characterized all our engineered promoters and found that the promoter activity decayed less under higher sucrose concentration conditions after replacing the constitutive promoter as well as multipling leader RNA (only in Pveg-2×LR promoter). Also, promoters with different numbers of leader RNA repeats showed different increases in activity after induction. In this part, the activity increase ~1.75 fold compared to the control. The promoter Pveg-5×LR show the highest(more than 2 fold) induction activity.

Fig 1 The characterization result of our engineered promoters

Biology

Pveg is an endogenous strong promoter of Bacillus subtilis, and the leader RNA sequence is from PsacB, which is a endogenous sucrose-sensitive promoter of Bacillus subtilis.The leader RNA sequence contains a terminator overlaping with the binding site of sacY [1] [2]. When the sucrose existed, the sacY can bind to the bing site in the leader RNA and stablized the terminator, which enables the transcription. The absence of sucrose can lead to the formation of terminator structure, which will lead to the early termination of downstream genes.

Reference

[1] Tortosa, P., & Le Coq, D. (1995). A ribonucleic antiterminator sequence (rat) and a distant palindrome are both involved in sucrose induction of the bacillus subtilis sacxy regulatory operon. Microbiology, 141(11), 2921–2927. https://doi.org/10.1099/13500872-141-11-2921

[2] Clerte, C., Declerck, N., & Margeat, E. (2013). Competitive folding of anti-terminator/terminator hairpins monitored by Single Molecule Fret. Nucleic Acids Research, 41(4), 2632–2643. https://doi.org/10.1093/nar/gks1315

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]