Week 10 HW: Advanced imaging & measurement technology
HTGAA 2026 — Week 10 Homework
Advanced imaging & measurement technology
Component Functions
E. coli Lysate
BL21 (DE3) Star Lysate (T7 RNA Polymerase-expressing)
Provides the endogenous transcription–translation machinery, including ribosomes, tRNAs, and metabolic enzymes. T7 RNA polymerase enables high-efficiency transcription from T7 promoters.
Salts and Buffer System
Potassium Glutamate
Try to maintains intracellular-like ionic conditions and stabilizes ribosomal complexes.HEPES-KOH (pH 7.5)
These buffers work on the system to maintain optimal enzymatic activity.Magnesium Glutamate
Critical cofactor for ribosome assembly and ATP-dependent processes; directly influences translation efficiency.Potassium Phosphate (Monobasic / Dibasic)
Provides buffering and supplies phosphate for metabolic and nucleotide regeneration processes.
Energy and Nucleotide System
Ribose
Precursor for nucleotide biosynthesis via salvage pathways.Glucose
Supports ATP regeneration through glycolysis.AMP, CMP, GMP, UMP
Nucleotide precursors that are phosphorylated into NTPs for transcription.Guanine
Salvageable base converted into GMP/GTP to sustain transcription.
Translation Substrate System
17 Amino Acid Mix
Supplies most amino acids required for translation.Tyrosine
Supplemented separately due to solubility constraints.Cysteine
Added independently due to oxidation sensitivity; critical for folding.
Additives
- Nicotinamide
Enhances NAD⁺ regeneration and supports metabolic activity.
Backfill
- Nuclease-Free Water
Adjusts reaction volume while preserving nucleic acid integrity.
System Comparison
1-Hour PEP-NTP System
- Rapid protein expression
- High initial yield
- Limited by substrate depletion and inhibitory byproducts
20-Hour NMP-Ribose-Glucose System
- Slower expression rate
- Sustained metabolic regeneration
- Improved stability and long-term output
Nucleotide Salvage Mechanism
Even without exogenous GMP, transcription proceeds via salvage pathways:
- Guanine → GMP → GTP
This maintains the nucleotide pool required for RNA synthesis.
Fluorescent Protein Considerations
| Protein | Key Property | Impact on CFPS |
|---|---|---|
| sfGFP | Fast folding | Early fluorescence readout |
| mRFP1 | Slow maturation | Delayed fluorescence |
| mKO2 | pH sensitivity | Performance depends on environment |
| mTurquoise2 | High quantum yield | Requires efficient folding |
| mScarlet-I | High brightness | Improved but slower maturation |
| Electra2 | Engineered variant | Dependent on folding and oxygen |
Design Hypothesis (36-Hour Optimization)
Target Protein: mRFP1
Strategy:
- Increase glucose and ribose concentrations
- Optimize Mg²⁺ levels
We enhanced energy regeneration and ribosome stability will support prolonged translation and improved folding, increasing fluorescence output over extended incubation.