5 days later tubes were assessed on paper colour to confirm survival.Īs Ducat stated limited growth is supported in Cyanobacteria as they use Hydrogen as reductant in alternative metabolic pathways. Tubes were flushed with nitrogen until air is replaced. 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.īlotted 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.įigure6. 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.įigure5. 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.įigure4. HydA and PsbA1 assembly gel resultsĪfter 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.įigure1: PsbA1 amplification gel results.Īmplified HydA and psbA1 genes were assembled via Polymerase Cycling Assembly (PCA) and also verified via gel electrophoresis as shown on Figure 3.įigure 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.
#Pasco capstone software manual
I will be happy to adapt this Lab Manual (for local publishing) and the associated computer software to accommodate your site, free of charge.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. In fact, student's conceptual foundation must be reinstalled. One cannot build new material on a faulty foundation.
Guide students through revisiting questions in step 1, and forming a new understanding. Confront students with hard experimental evidence of the true behavior of nature 3. If you don't do this, in step 3 students will misremember what they originally thought. These should be representations of what they really "feel" about a phenomenon. Ask students questions about possible misconceptions - free-association. It also employs the CCD model to eradicate misconceptions about how nature works. It has been upgraded to accommodate PASCO Capstone Software. It has been tested with the FCI, and produces maximum gain found in literature. These should be re This is an Introductory Physics Term I Laboratory Manual.
This is an Introductory Physics Term I Laboratory Manual.