It was soaked in the medium with sulfide and w/o sulfide. From Figure 7, it can be seen that transformed HydA+ cells differ significantly from the wild-type by its color illustrating the higher survival rate compared to the wild-type strains.Д«lotted paper was also assessed using survival assay with the same condition only few adjustments for the paper. The samples of nearly same OD were constantly shaken in the shaking incubator. The effect of genetic modification on survivability of cyanobacteria in a hydrogen sulfide (H2S) rich and anaerobic environment was assessed qualitatively. HydA+PsbA1 amplification from the construct integrated into cyanobacteria Out of five samples, the 5th band without ladder had an expected band for ~3.1 kb.ДЇigure6. NSb primer was used due to complications during amplification process with HydA primers. To verify the integration of construct into cyanobacteria psbA1+hydA(~3.1 kb) amplification was performed using forward psbA1 and reverse NSb primers. As a negative control p233 (also known as MF) without insert was used which did not show any band for HydA as expected.ДЇigure5. Overall, four samples were assessed for the HydA and three were positive for the gene as bands corresponding to ~1.7-1.8 kb were visualized on the gel. To ensure the presence of HydA gene in the construct, the samples were linearized by EcoRV and used as template for HydA amplification (see Figure 5). Linear assembled construct digested by BamH gel results All samples were linearized with BamHI-HF beforehand.ДЇigure4.

HydA and PsbA1 assembly gel resultsДЄfter transformation of the assembled construct into the DH5alpha cloning strain, DNA extraction results illustrated bands higher than empty vector p233 (also known as MF (Maturation Factor)). As it can be seen from Figure 1 and Figure 2 successful amplification of genes of interest was verified via gel electrophoresis.ДЇigure1: PsbA1 amplification gel results.ДЄmplified HydA and psbA1 genes were assembled via Polymerase Cycling Assembly (PCA) and also verified via gel electrophoresis as shown on Figure 3.ДЇigure 3. To assemble psbA1 and HydA genes, firstly, amplification of psbA1 (503 bp) from p233(11830 bp) and HydA(1998 bp) from plasmid PAM (5004 bp) were done.

Plasmid p233 that contains hydrogenase maturation factors HydEF and HydG under psbA1 promoter and HydA under psbA1 promoter in 0015 plasmids. Due to complications with amplification and assembly of synthetic sequences of genes that we ordered like complex stable secondary structures and a lot of long repeats we switched to using plasmids that we received from as a kind gesture from Daniel C.

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