Difference between revisions of "Part:BBa K823002"

Revision as of 11:49, 22 September 2012

P_lepA

P_lepA is the promoter of the lepA gene of Bacillus subtilis. It is a constitutive promoter and does not contain a ribosome binding site.

Usage and Biology

P_lepA is constitutive promoter which is important for the transcription of a bicistronic operon. One of the expressed proteins is the protein PlepA [http://www.ncbi.nlm.nih.gov/pubmed?term=Microbiology%2C%20142%3A%201641%E2%80%931649: (Homuth et al., 1996)]. This protein plays an important role during the translation as it can move the mRNA-tRNA complex one step back in the ribosome which is expected to improve the fidelity of translation [http://www.ncbi.nlm.nih.gov/pubmed?term=Cell%2C%20127%20%284%29%3A%20721%E2%80%93733: (Qin et al., 2006)]. This promoter was evaluated with the lux operon as a reporter. For more Details visit the [http://2012.igem.org/Team:LMU-Munich/Data Data] page of the LMU-Munich Team 2012 .

Evaluation

Luminescence measurement of the constitutive Bacillus promoters P_liaG and P_lepA in the reporter vector pSB_Bs3C-luxABCDE. OD₆₀₀ (up), LUMI (middle) and LUMI per OD₆₀₀ (down) depending on the time (h) are shown for two different clones (green/blue). Data derive from three independent experiments, the graphs show the mean fo the three experiments and the standard deviation. Curves were fitted over each other (t=0, OD₆₀₀=0,3) and smoothed by taking the average of three neighboring values.

The constitutive promoters P_liaG and P_lepA were evaluated with the lux operon as a reporter. This is why the promoter activity leads to gene expression and to the production of the protein luciferase. The luminescence of this protein can be measured with the plate reader Synergy2 (Biotek). All clones show a normal growth behaviour. The activity of the promoters increases during transition from log to stationary phase. The second clone of the promoters P_lepA and P_liaG did not show any luminescence activity. Therefore additional clones should be measured. In the beginning of the growth curve the activity of both promoters increases to their maximum. This maximum activity appears during the same growth phase. P_liaG has an activity maximum of about 100.000 Lumi/OD₆₀₀ during the transition from logarithmic to the stationary phase. P_lepA shows a maximum of about 400.000 Lumi/OD₆₀₀. Comparing these two constitutive promoters the activity of P_lepA is about four times higher than the activity of P_liaG. In the late stationary phase the activity completely disappears.

Sequence and Features

This part was amplified from the genom of B. subtilis.