Evaluating the effect of promoter strength on the complementation of deletion mutants in Synechocystis sp. PCC 6803

The CO2 uptake mechanism within Cyanobacteria plays a vital role in our environment due to its photosynthetic ability to uptake CO2 and produce oxygen. Cyanobacteria possesses an inorganic carbon concentrating mechanism including two CO2 specific uptake systems. One is constitutive and one is low-CO2 inducible. By limiting the CO2 availability, the bacteria will express the inducible CO2 concentrating mechanisms to survive. These systems include NDH-13, the inducible complex and NDH-14, the constitutive complex. The goal of our lab is to focus our studies on the inducible NDH-13 system, however, there are concerns that the promoter strength of our complementation strains will prove problematic to future studies. To overcome this concern, we tested three different promoters with varied expression patterns and then determined which would yield the best construct for future experiments. Included were the constitutive Rubisco and PsbA2 promoters, along with the native NdhF3 promoter. Using Gibson Assembly, plasmids containing these variations were constructed. A deletion mutant was also constructed within our lab lacking both systems, C2. Spot assays on BG-11 media were completed under conditions of air and CO2 gassing. A positive, wild-type, control and negative, M55, control was tested along with C2 and the three complementations. A phenotypic analysis was completed to observe if the promoter strength influences the cells' ability to grow within the different conditions.Lew Wentz FoundationMicrobiology and Molecular Genetic