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Background  Cyanobacteria are solar-powered prokaryotes useful for sustainable production of valuable molecules, but orthogonal and regulated promoters are lacking. The Lac repressor (LacI) from  Escherichia coli  is a well-studied transcription factor that is orthogonal to cyanobacteria and represses transcription by binding a primary  lac  operator ( lacO ), blocking RNA-polymerase. Repression can be enhanced through DNA-looping, when a LacI-tetramer binds two spatially separated  lacO  and loops the DNA. P trc  is a commonly used LacI-repressed promoter that is inefficiently repressed in the cyanobacterium  Synechocystis  PCC 6803. P trc 2O, a version of P trc  with two  lacO , is more efficiently repressed, indicating DNA-looping. To investigate the inefficient repression of P trc  and cyanobacterial DNA-looping, we designed a P trc -derived promoter library consisting of single  lacO  promoters, including a version of P trc  with a stronger  lacO  (Ptrc1O-proximal), and dual  lacO  promoters with varying inter- lacO  distances (the P trc 2O-library).    Results  We first characterized artificial constitutive promoters and used one for engineering a LacI - expressing strain of  Synechocystis . Using this strain, we observed that P trc 1O-proximal is similar to P trc  in being inefficiently repressed. Further, the P trc 2O-library displays a periodic repression pattern that remains for both non- and induced conditions and decreases with longer inter- lacO  distances, in both  E. coli  and  Synechocystis . Repression of P trc 2O-library promoters with operators out of phase is less efficient in  Synechocystis  than in  E. coli , whereas repression of promoters with  lacO  in phase is efficient even under induced conditions in  Synechocystis . Two well-repressed P trc 2O promoters were highly active when tested in absence of LacI in  Synechocystis .    Conclusions  The artificial constitutive promoters herein characterized can be utilized for expression in cyanobacteria, as demonstrated for LacI. The inefficient repression of P trc  and P trc 1O-proximal in  Synechocystis , as compared to  E. coli , may be due to insufficient LacI expression, or differences in RNAP subunits. DNA-looping works as a transcriptional regulation mechanism similarly as in  E. coli . DNA-looping contributes strongly to P trc 2O-library repression in  Synechocystis , even though they contain the weakly-repressed primary  lacO  of P trc 1O-proximal and relatively low levels of LacI/cell. Hence,  Synechocystis  RNAP may be more sensitive to DNA-looping than  E. coli  RNAP, and/or the chromatin torsion resistance could be lower. Two strong and highly repressed P trc 2O promoters could be used without induction, or together with an unstable LacI.

Design and analysis of LacI-repressed promoters and DNA-looping in a cyanobacterium