This section outlines our experimental journey and key learnings during the iGEM project cycle, focusing on strain verification, gene cassette construction, transformation, protein expression, growth optimization, and carbon fixation analysis.
To confirm gene integration, PCR was performed on constructed yeast strains containing RuBisCO Form I and II genes using Kit 5 and Kit 6 as references. PCR results exhibited inconsistency, with amplification success varying despite identical conditions. To mitigate this, we reduced reaction volumes and increased parallel PCR replicates, improving overall success rate.
The target DNA fragment was ligated into Kit 1 using restriction enzymes XhoI and NotI. Initial ligation attempts failed, as PCR verification did not yield expected fragment sizes. After performing an enzyme activity assay, low enzyme activity was confirmed and enzymes were replaced with fresh ones, resulting in successful ligation.
Variations in gel extraction and purification led to inconsistent DNA purity affecting electroporation success. Following recovery and plating on G418-containing YPD agar, initial PCR verification failed to detect target bands. Repeating gel purification to improve DNA quality followed by electroporation enabled successful integration confirmed by PCR.
Cultivation and Antibiotic Selection: Multiple antibiotic concentrations were used during plating to minimize selection bias and improve strain validation accuracy, due to variable antibiotic tolerance among clones.
Protein Expression Confirmation: SDS-PAGE failed to detect expected A2 β-casein bands, likely due to limited cell disruption from freeze–thaw methods. Future plans include optimizing lysis methods and introducing GFP reporters to verify gene expression and locate issues.
OD600 Growth Curve Measurement: Large yeast cell size caused sedimentation bias, affecting OD sampling. Switching to pipette volumes equal to sample volume for resuspension improved measurement consistency and reproducibility.
Gas Chromatography Analysis: Initial GC measurements over 48 hours in triplicates revealed oxygen depletion after 24 hours and lacked correlation with growth phase due to absent cell growth monitoring. Also, sequential strain measurements risked sample contamination; syringes sealed with rubber stoppers showed minor air leaks affecting accuracy. Later experiments improved by collecting gas and liquid samples every 4 hours for 24 hours with single replicates to reduce variability.
Planned work includes extending sampling duration and studying RuBisCO’s function and regulation under anaerobic conditions to understand its impact on yeast growth and carbon metabolism.