Difference between revisions of "Part:BBa K823002"

Latest revision as of 16:56, 3 February 2014

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 the bicistronic operon. One of the expressed proteins is the protein P_lepA [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/Constitutive Data] page of the LMU-Munich Team 2012 or get an overview of our whole project [http://2012.igem.org/Team:LMU-Munich Beadzillus].

Evaluation

This part was also evaluated in the publication [http://www.jbioleng.org/content/7/1/29 The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis] by Radeck et al..

Luminescence measurements

The constitutive promoters P_liaG and P_lepA were evaluated in the reporter vector pSB_Bs3C-luxABCDE which contains the lux operon. The promoter activity leads to gene expression and to the production of the protein luciferase. The luminescence produced by this protein can be measured with the plate reader Synergy2 (Biotek) (Fig.1).

Fig. 1: Luminescence measurement of the constitutive Bacillus promoters PliaG and PlepA in the reporter vector pSBBs3C-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.

All clones show a normal growth behaviour. The activity of both promoters increases during transition from log to stationary phase. P_liaG has an activity maximum of about 100.000 Lumi/OD₆₀₀. 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. The second clone of the promoters P_lepA and P_liaG did not show any luminescence activity. Therefore additional clones should be measured.

β-galactosidase assay

The two constitutive promoters P_liaG and P_veg were evaluated with the reporter vector pSB_Bs1C-lacZ which contains the lacZ reporter gene (Fig.2).

Fig. 2: β-galactosidase assay and growth curve of strains carrying the promoters PliaG (black) and Pveg (grey) fused to lacZ. β-galactosidase activity (Miller Units) and the growth curve values are the average of two independent clones with their standard deviation. Experiment shows representative data from three independent experiments.

Promoter activity leads to the expression of the β-galactosidase. The β-galactosidase assay of the constitutive Bacillus promoters P_veg and P_liaG was repeated three times. The graph shows data of one representative experiment. In the beginning of the growth curve both promoters show only low activity before it increases to a maximum before it decreases to the begininng level after about seven hours (Data not shown). Summing up, the course of activity of both promoters P_veg and P_liaG is very similar based on the growth curve. The highest β-galactosidase activity and therefore the highest activity of the promoter P_veg with a maximum of 65 Miller units can be found during the transition from the logarithmic to the stationary phase. This is about five times higher than the acitivity of the promoter P_liaG with a maximum activity of about 12 Miller Units.

Sequence and Features

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