Week 12 –Bioproduction & Cloud Labs
Week 12 HW: Bioproduction
sfGFP
- sfGFP folds extremely efficiently and matures quickly, even at lower temperatures, making it highly reliable in cell‑free systems.
- Its chromophore formation still requires oxygen, so fluorescence can lag if oxygen becomes limiting in sealed reactions.
mRFP1
- mRFP1 has a slower chromophore maturation rate than GFP variants, delaying fluorescence onset in cell‑free expression.
- It also folds less efficiently, making its brightness more sensitive to temperature and chaperone availability.
mKO2
- mKO2 tends to mature more slowly and is more prone to misfolding or aggregation, which can reduce yield in cell‑free systems.
- Orange FPs often show increased sensitivity to ionic strength and temperature during folding.
mTurquoise2
- mTurquoise2 is very bright but its fluorescence is sensitive to pH and Mg²⁺/ionic conditions, which can shift quantum yield in cell‑free reactions.
- It also requires efficient folding to maintain its high quantum yield, making it sensitive to crowding and chaperone levels.
mScarlet‑I
- mScarlet‑I is one of the fastest‑maturing red FPs but still requires optimal folding conditions to reach full brightness.
- Red FPs are generally more temperature‑dependent, so suboptimal incubation conditions can reduce the fraction of properly folded protein.
Electra2
- Electra2 is engineered for rapid maturation and high brightness but still depends on oxygen availability for chromophore formation.
- Its fluorescence output can drop if pH drifts acidic during long incubations.
Hypothesis for Improving Fluorescence Over 36 Hours
Example Hypothesis
Protein: mScarlet‑I
Reagent Adjustment: Add supplemental chaperones (e.g., GroEL/ES) and increase PEG‑8000 concentration slightly in the 2× master mix.
Expected Effect: Enhanced folding efficiency and reduced aggregation will increase the proportion of properly folded, fluorescent mScarlet‑I, maximizing red fluorescence over the 36‑hour incubation.
Additional Optional Hypotheses
Protein: mTurquoise2
Reagent Adjustment: Increase HEPES buffer concentration and adjust Mg²⁺/K⁺ levels to stabilize pH and ionic environment.
Expected Effect: Stabilizing pH and ionic strength will preserve mTurquoise2’s high quantum yield, increasing overall cyan fluorescence.
Protein: sfGFP / Electra2
Reagent Adjustment: Increase buffer capacity and incorporate an oxygen‑enhancing strategy (e.g., higher surface‑to‑volume ratio or mild oxidizing cofactor).
Expected Effect: Sustained neutral pH and improved oxygen availability will support continuous chromophore maturation, maximizing green fluorescence output.