Week 11: Bioproduction & Cloud Labs

The 1,536 Pixel Artwork Canvas | Collective Artwork

While I don’t exactly remember what I did in the artwork, since I forgot to note it down, I remember making 2-10 edits in the artwork. It took me a little too much time to understand what was exactly happening, but I loved the whole project and the experience of doing it.

Cell-Free Protein Synthesis | Cell-Free Reagents

1. Components in Cell-free systems and their uses

E. coli Lysate

• BL21 (DE3) Star Lysate (includes T7 RNA Polymerase): This supplies the raw translation machinery like ribosomes, tRNAs, and initiation factors needed to synthesize protein from mRNA. Since it has T7 RNA Polymerase built into it, it can drive both transcription and translation simultaneously in a single tube.

Salts/Buffer

• Potassium Glutamate: Serves as the main salt in the reaction to keep the ionic strength at physiological levels, which helps stabilize the ribosomes during elongation.
• HEPES-KOH pH 7.5: It is a zwitterionic buffer that holds the reaction mix at a stable, neutral pH so the metabolic enzymes don’t denature over time.
• Magnesium Glutamate: Supplies the essential Mg2+ ions required as cofactors for proper ribosome assembly and enzyme function.
• Potassium phosphate monobasic / Potassium phosphate dibasic: Works as a secondary buffering system to control pH while maintaining a steady pool of inorganic phosphate for nucleotide recycling.

Energy / Nucleotide System

• Ribose: A core sugar precursor that enters the pentose phosphate pathway to help the system synthesize its own nucleotide backbones sustainably.
• Glucose: The primary carbon source that feeds into glycolysis, generating the ATP and GTP needed to keep fueling the transcription and translation machinery.
• AMP / CMP / UMP: These are the monophosphate nucleotide precursors (NMPs) that get phosphorylated into their active triphosphate forms (ATP, CTP, UTP) for RNA synthesis.
• GMP: The monophosphate precursor for GTP, which is kept at 0 uM here because the system is designed to build it from the free base instead.
• Guanine: A free purine nucleobase that the salvage pathways in the lysate can use to build the guanosine nucleotide pool from scratch.

Translation Mix (Amino Acids)

• 17 Amino Acid Mix: Provides the standard structural building blocks required for peptide chain elongation.
• Tyrosine: Supplemented separately because it has poor solubility at neutral pH and needs to be prepared at pH 12 to ensure it doesn’t precipitate out.
• Cysteine: Added on its own because it degrades and oxidizes very quickly in cell-free environments, so it needs to be carefully topped up.

Additives

• Nicotinamide: Helps prevent the breakdown of crucial NAD+ cofactors, keeping the metabolic energy pathways running stably during long runs.

Backfill

• Nuclease Free Water: Used to top the master mix to the final volume while ensuring there are no RNAses or DNAses around to cleave the DNA/RNA templates.

2. Describe the main differences between the 1-hour optimized PEP-NTP master mix and the 20-hour NMP-Ribose-Glucose master mix shown in the Google Slide above. (2-3 sentences)

The 1-hour mix is a PEP/NTP-based setup designed for an immediate, high-energy burst by supplying fully formed triphosphates (ATP, GTP, etc.) and phosphoenolpyruvate, which burns out quickly. On the other hand, the 20-hour mix utilizes an NMP-Ribose-Glucose framework that relies on cheap, simple precursors instead of pre-made energy molecules. This allows the native enzymes remaining in the lysate to continuously recycle and synthesize nucleotides in situ, making it a more sustainable system for long, extended incubations like making artwork.

3. How can transcription occur if GMP is not included but Guanine is?

Transcription can still happen because the E. coli lysate contains functional, endogenous purine salvage pathway enzymes (like purine phosphoribosyltransferase). This enzyme takes the free guanine base and attaches a phosphoribosyl group to it, converting it directly into GMP inside the tube. From here, standard native kinases sequentially phosphorylate that GMP into GDP and then GTP, providing the building blocks the T7 RNA Polymerase needs for transcription.

Planning the Global Experiment | Cell-Free Master Mix Design

1. Given the 6 fluorescent proteins we used for our collaborative painting, identify and explain at least one biophysical or functional property of each protein that affects expression or readout in cell-free systems. (Hint: options include maturation time, acid sensitivity, folding, oxygen dependence, etc) (1-2 sentences each) sfGFP mRFP1 mKO2 mTurquoise2 mScarlet_I
   Electra2 The amino acid sequences are shown in the HTGAA Cell-Free Benchling folder.

Protein Properties: Acid Sensitivity (pKa) The pKa value represents the pH at which 50% of the protein’s fluorescence is quenched. In cell-free systems, the pH often drops over time as metabolic byproducts accumulate, making this a critical factor for long-term readouts.

• sfGFP: With a pKa of approximately 6.0, sfGFP is remarkably robust; its rapid folding and high stability mean it remains bright even if the reaction environment becomes slightly acidic over the 36-hour incubation.
• mRFP1: This protein has a pKa near 4.5, making it highly resistant to acid quenching; however, its relatively slow maturation compared to newer reds means the readout might lag behind its actual expression levels.
• mKO2: With a pKa around 5.0, mKO2 is quite stable in varying pH levels, ensuring that its orange fluorescence remains consistent even as the master mix’s buffering capacity is tested.
• mTurquoise2: This variant has a pKa of 3.1, making it one of the most acid-tolerant proteins in your set; it is virtually immune to quenching caused by the pH shifts typical of long-duration cell-free reactions.
• mScarlet-I: It has a pKa of 5.4, which is relatively good for a bright red FP, though it is more sensitive than mTurquoise2; its high brightness is its main advantage, but it may dim if the pH drops significantly below 6.0.
• Electra2: As a fast-maturing variant, its pKa is generally designed to be stable (around 5.0); its primary functional property is its rapid signal appearance, which must be balanced against the gradual acidification of a 36-hour reaction.

2. Create a hypothesis for how adjusting one or more reagents in the cell-free mastermix could improve a specific biophysical or functional property you identified above, in order to maximize fluorescence over a 36-hour incubation. Clearly state the protein, the reagent(s), and the expected effect.

Protein: mScarlet-I

Reagent(s): HEPES-KOH and Magnesium Glutamate

Hypothesis: Increasing HEPES-KOH to 100 mM and Magnesium Glutamate to 10 mM will maximize mScarlet-I fluorescence. Higher buffering capacity prevents the pH from falling toward the pKa of 5.4 as acidic metabolic byproducts accumulate, while additional magnesium compensates for the ions that are often chelated by those same byproducts over a 36-hour period.

3. The second phase of this lab will be to define the precise reagent concentrations for your cell-free experiment. You will be assigned artwork wells with specific fluorescent proteins and receive an email with instructions this week (by April 24). You can begin composing master mix compositions here.

4. The final phase of this lab will be analyzing the fluorescence data we collect to determine whether we can draw any conclusions about favorable reagent compositions for our fluorescent proteins. This will be due a week after the data is returned (date TBD!). The reaction composition for each well will be as follows: 6 μL of Lysate 10 μL of 2X Optimized Master Mix from above 2 μL of assigned fluorescent protein DNA template 2 μL of your custom reagent supplements Total: 20 μL reaction

Thank You!