The carbon fixation mechanism of cyanobacteria was thoroughly studied, from the steps and molecules involved in the importation of CO2 into the cytoplasm and the center of reaction, the carboxysome, up to the rate of the enzymatic reactions and the regulatory mechanisms that determine how much CO2 is our cyanobacteria is able to harness. The bottlenecks of this process were found to be present in two key steps: in the direct incorporation of CO2 gas into ribulose-1,5-biphosphate (RuBP) by the enzyme RubisCO due to lack of specificity to the substrate and slow rate of reaction, and the subsequent CO2 gas leakage out of the carboxysome to the cytoplasm expulsion onto the extracellular media.
Three alternatives of solution were explored to increase the carbon fixation capacity of Synechococcus sp., these alternatives revolved around the availability of substrate to the enzyme, the regulatory mechanisms involved in the expression of the genes related to carbon fixation, and the catalytic activity of the enzymes involved in this process. Following this logic, designed our experimental strategy to attack and increase the efficiency of the process. First, light control was chosen as a smart and easy way to regulate the gene expression (BBa_K754000), then a construct to modify the importation mechanism of CO2 into the cell was designed (BBa_K3106006), and lastly a construct to increase the intracellular concentration of RubisCO was designed (BBa_K3106008). Another construct was designed to test if both steps of the pathway were modified (BBa_K3106009).