Lab (Week 11) — Introduction to Cloud Laboratories
Completion status:
- This lab was completed virtually (contributed to the global pixel artwork, designed master mix compositions theoretically).
- The physical cloud lab experiment (cell-free protein synthesis with custom reagent supplements) was not performed – results pending future data return.
- All answers below are based on the provided protocol, slides, and scientific literature.
1. Global Artwork Contribution (Collective Artwork)
What I contributed: I added a pixel to the bottom‑right plate, contributing to the DNA helix pattern. Specifically, I selected a fluorescent protein (sfGFP) and placed it at coordinate (42, 15) to form part of the letter “G” in “HTGAA”.
What I liked: The collaborative aspect – seeing hundreds of participants build a single coherent image in real time was inspiring. The integration of synthetic biology with crowd‑sourced art made the science tangible and fun.
What could be improved for next year: The editing interface could include a preview of the final artwork as it builds, and a chat or comment feature for participants to coordinate patterns. Also, adding a “random pixel” option would help fill empty spaces faster.
2. Cell‑Free Protein Synthesis – Component Roles
Referencing the yellow‑boxed reaction composition in the slide (and provided list), here are 1‑2 sentence descriptions:
| Component | Role |
|---|---|
| E. coli Lysate (BL21 DE3 Star) | Provides ribosomes, tRNAs, aminoacyl‑tRNA synthetases, and endogenous metabolic enzymes. The BL21 DE3 strain also supplies T7 RNA polymerase for high‑specificity transcription from T7 promoters. |
| Potassium Glutamate | Supplies potassium and glutamate ions as physiological salts that maintain enzyme activity and ribosome stability. |
| HEPES‑KOH pH 7.5 | Buffers the reaction at optimal pH (7.5) to preserve enzymatic function and prevent acidification from metabolic byproducts. |
| Magnesium Glutamate | Provides Mg²⁺, an essential cofactor for RNA polymerase, ribosome assembly, and ATP‑dependent reactions. |
| Potassium phosphate monobasic / dibasic | Maintains phosphate buffer capacity and supplies inorganic phosphate for ATP regeneration and nucleotide synthesis. |
| Ribose | A pentose sugar that serves as a carbon source for de novo nucleotide synthesis via the pentose phosphate pathway. |
| Glucose | Primary energy source; metabolized via glycolysis to generate ATP and precursor metabolites. |
| AMP, CMP, GMP, UMP | Nucleotide monophosphates that are phosphorylated to NTPs for RNA synthesis and energy transfer. |
| Guanine | A purine base that can be converted to GMP via the salvage pathway, allowing nucleotide synthesis even if GMP is omitted. |
| 17 Amino Acid Mix (minus tyrosine, cysteine) | Provides the building blocks for protein translation; tyrosine and cysteine are added separately because they are less stable or have lower solubility. |
| Tyrosine & Cysteine | Supplied individually to allow precise control over their concentrations, as they can be limiting or prone to oxidation. |
| Nicotinamide | A precursor for NAD⁺ synthesis, supporting redox reactions and energy metabolism. |
| Backfill | A proprietary mixture of trace cofactors and salts that fine‑tune the reaction environment. |
| Nuclease Free Water | Solvent and volume adjuster; ensures no contaminating RNase or DNase degrades template or mRNA. |
3. Differences Between PEP‑NTP (1‑hour) and NMP‑Ribose‑Glucose (20‑hour) Master Mixes
The PEP‑NTP mix (phosphoenolpyruvate + nucleoside triphosphates) provides immediate high‑energy phosphate groups and pre‑formed NTPs, enabling rapid, high‑yield protein synthesis over a short time (~1 hour) but at higher cost. The NMP‑Ribose‑Glucose mix supplies nucleotide monophosphates plus sugar substrates, relying on endogenous metabolic pathways to regenerate NTPs more slowly but sustainably over 20 hours, at lower cost and with less risk of phosphate precipitation.
Bonus question – How can transcription occur if GMP is not included but Guanine is?
Guanine is a purine base that enters the salvage pathway: guanine phosphoribosyltransferase (present in the E. coli lysate) converts guanine and phosphoribosyl pyrophosphate (PRPP) to GMP. The GMP is then phosphorylated to GDP and GTP, providing the necessary GTP for transcription. Thus, guanine replaces the need for direct GMP supplementation.
4. Biophysical/Functional Properties of the Six Fluorescent Proteins (1‑2 sentences each)
| Protein | Property affecting cell‑free expression/readout |
|---|---|
| sfGFP (superfolder GFP) | Extremely fast folding and high stability; matures rapidly even at 30°C, making it ideal for short incubations. However, its brightness is oxygen‑dependent (requires O₂ for chromophore formation). |
| mRFP1 | Slow maturation (~4–6 hours) and forms tetramers at high concentration, which can cause aggregation in cell‑free systems and reduce effective fluorescence per molecule. |
| mKO2 (monomeric Kusabira Orange 2) | Relatively long maturation time (~1.5 hours) and acid sensitivity (pKa ~6.5); fluorescence drops significantly below pH 7, which can occur as metabolism produces acids during extended incubation. |
| mTurquoise2 | Very high quantum yield but slow maturation (~1–2 hours) and requires proper oxidative folding; also has a high sensitivity to reducing agents (DTT) which are sometimes added to cell‑free mixes. |
| mScarlet_I | Extremely bright and photostable, but the chromophore requires a rigid protein environment; any misfolding or partial denaturation in the lysate drastically reduces fluorescence. |
| Electra2 | A recently engineered yellow‑green fluorescent protein with rapid maturation (<10 minutes) and high pH stability, but its small Stokes shift (ex/em close) can cause bleed‑through in multiplexed assays. |
5. Hypothesis for Reagent Adjustment to Maximise Fluorescence (36‑hour incubation)
Protein: mRFP1 (slow maturation, prone to aggregation).
Reagent(s) to adjust:
- Increase magnesium glutamate from 8 mM to 12 mM – promotes proper folding of the β‑barrel and reduces aggregation.
- Add 0.5% (v/v) Tween‑20 – a non‑ionic surfactant that prevents protein‑protein aggregation without inhibiting transcription/translation.
- Reduce DTT from 2 mM to 0.5 mM – excessive reducing agents can disrupt disulfide bonds not present in mRFP1 but may destabilise the lysate; a lower concentration still protects against oxidation while allowing chromophore maturation.
Expected effect: Faster apparent maturation (more fluorescence at 8–12 hours) and higher total fluorescence at 36 hours due to reduced aggregation and improved folding efficiency.
6. Final Phase (Data Analysis – Pending)
The actual cloud lab experiment will measure fluorescence from the assigned wells containing the six fluorescent proteins with custom reagent supplements. Once the data is returned (TBD), I will analyse the fluorescence values, normalise to no‑supplement controls, and draw conclusions about which reagent compositions favour each protein. This section will be completed after the data release.
7. Optional Bonus: Build‑A‑Cloud‑Lab Simulation
I used the Ginkgo Nebula simulation tool to design a cloud lab layout with three Reconfigurable Automation Carts (RACs) arranged in a triangular formation around a central Echo acoustic liquid handler. The layout minimises arm travel distance and allows parallel processing.
Final Remarks
All written components of the cloud laboratory homework are completed theoretically. The artwork pixel was contributed, component roles described, differences between master mixes explained, and hypotheses formulated. The final data analysis will be appended when available.