The cultures have been transformed with a self replicative vector, pSUN202, where truncated versions of the hupSL promoter have been fused to gfp (Selleck ABT 263 constructs A to E).

Dilutions of the cultures, ranging from 3–30 μg Chl a/ml, have been plotted against the intensity (%). All dilutions have been measured in triplicates and the total fluorescence in the sample is 100%. Generation of hupSL reporter gene constructs To define and identify selleckchem regulatory regions in the promoter controlling hupSL transcription a deletion analysis of the promoter was carried out. Five hupSL promoter sequences of various lengths (A-E; Fig. 1) were cloned by PCR and coupled to gfp, encoding the reporter protein GFP, or to luxAB encoding the reporter enzyme Luciferase (Fig. 1). The lengths of the truncated promoter fragments were designed according to the positions of the putative binding sites for Integration Host Factor (IHF) and NtcA, identified in the hupSL

promoter using bioinformatics (Fig. 1) [14]. Confirmation of the insertion of correct promoter deletions constructs Cells from N. punctiforme were transformed by electroporation with vector constructs containing various lengths of the hupSL promoter coupled to gfp (A-E) or luxAB (1–5) (Fig. 1). Positive clones were confirmed by colony PCR. The primers used for the colony PCR anneal to the vector sequences flanking the inserted promoter region and hence the product spans the full length of the insert (Table 1). Analysis of the obtained results indicates that all the cloned fragments were of www.selleckchem.com/products/XL880(GSK1363089,EXEL-2880).html a length expected for the correct construct (data not shown). Optimization of GFP fluorescence measurements To be able to compare the GFP

expression from the different promoter deletions, dilution series were made to confirm that measurements were done in a range where the GFP signal are linear for all the constructs. The curves show high R2 values, ranging between 0.96 to 1.0, confirming that there is only very little or no saturation of the signal using the cell density chosen for the measurements (assessed by Chlorophyll a concentration) (Fig. 3). Experiments with dilution series Fludarabine order of the bioluminescence measurements showed high R2 values ranging from 0.79 – 0.99 Expression from the hupSL promoter deletions The measurements of GFP intensity and hence promoter activity were performed on living cells grown under nitrogen fixing conditions. The shortest promoter fragment, E, (stretching from -57 to tsp), showed the highest expression level (Fig. 4) in all experiments. This was also confirmed in the measurements of bioluminescence, where construct E showed the highest expression levels (data not shown). This part of the hupSL promoter lacks the putative IHF and NtcA binding sites (Fig. 1). There were minor variations between the promoter activities of the four longer promoter fragments (construct A-D).