Week 11 HW: Building Genomes

Part B: Cell-Free Protein Synthesis | Cell-Free Reagents

1. Referencing the cell-free protein synthesis reaction composition (the middle box outlined in yellow on the image above, also listed below), provide a 1-2 sentence description of what each component’s role is in the cell-free reaction.

   • BL21 (DE3) Star lysate (includes T7 RNA polymerase): This provides the core cellular machinery needed for protein synthesis, incl~uding ribosomes, tRNAs, enzymes, and other translation factors. Because it includes T7 RNA polymerase, it can also transcribe DNA templates with a T7 promoter into mRNA.

   • Potassium glutamate: This helps maintain the ionic environment needed for ribosomes and enzymes to work properly. It supports protein synthesis by mimicking intracellular salt conditions.

   • HEPES-KOH pH 7.5: This acts as a buffer to keep the reaction at a stable pH. A stable pH is important because transcription and translation enzymes are sensitive to pH changes.

   • Magnesium glutamate: Magnesium is essential for ribosome function, RNA stability, and many enzyme reactions in transcription and translation. If magnesium is too low or too high, protein production drops.

   • Potassium phosphate monobasic: This helps buffer the reaction and contributes phosphate needed in the overall chemical environment. It also helps maintain proper ionic strength.

   • Potassium phosphate dibasic: This works together with monobasic phosphate to maintain the phosphate buffer system. Together they help keep the reaction chemically stable.

   • Ribose: Ribose serves as part of the energy regeneration system and can help support longer-lasting reactions. It provides carbon that can feed metabolic pathways in the lysate.

   • Glucose: Glucose is another energy source that helps regenerate ATP through metabolism in the extract. This is important for sustaining transcription and translation over time.

   • AMP: AMP is part of the nucleotide and energy balance in the reaction. It can be recycled through the lysate’s metabolic pathways to help support ATP regeneration.

   • CMP: CMP is a pyrimidine nucleotide precursor that supports RNA synthesis and nucleotide recycling. It helps maintain the pool of RNA building blocks.

   • GMP: GMP is a guanine nucleotide precursor that supports RNA synthesis and nucleotide recycling. It contributes to making the GTP needed for transcription and translation.

   • UMP: UMP is another RNA nucleotide precursor that supports transcription and nucleotide balance. It helps maintain enough pyrimidine nucleotides in the reaction.

   • Guanine: Guanine can be salvaged by enzymes in the lysate and converted into guanine nucleotides such as GMP and GTP. This helps support transcription even if GMP is not added directly in high amounts.

   • 17 amino acid mix: This provides most of the amino acids needed to build proteins during translation. They are the basic building blocks for the new protein.

   • Tyrosine: Tyrosine is added separately because it may be less stable or less soluble in the standard mix. It is still required as one of the amino acids for protein synthesis.

   • Cysteine: Cysteine is also added separately because it is more chemically sensitive than many other amino acids. It is needed for protein synthesis and for forming disulfide-containing proteins if relevant.

   • Nicotinamide: Nicotinamide supports metabolic enzyme activity by helping maintain redox cofactor pools such as NAD-related systems. This can improve energy regeneration and reaction longevity.

   • Nuclease-free water: This is used to bring the reaction to the correct final volume and concentration. It is nuclease-free so the DNA and RNA are not degraded.

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 main difference is cost and how the system generates usable nucleotides. The 1-hour PEP-NTP mix directly adds the more expensive NTPs, while the 20-hour NMP-ribose mix uses cheaper NMPs plus ribose/glucose and a longer incubation time so the reaction can regenerate the needed NTPs inside the mix itself.

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

   • Transcription can still occur because guanine can be converted by nucleotide salvage enzymes in the lysate into GMP, and then further into GDP and GTP. In other words, the extract can recycle guanine into the usable nucleotide form needed by RNA polymerase.

Part C: 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 sfGFP is a good cell-free reporter because it was engineered to fold very robustly, so it is less likely to lose signal from misfolding than standard GFPs. It also matures relatively fast, with FPbase listing a maturation time of about 13.6 min, so fluorescence appears fairly quickly after translation.
   • mRFP1 A key limitation of mRFP1 in cell-free systems is its slow maturation: FPbase lists it at about 60 min, so the protein can be made before the red signal is fully visible. It also has a relatively low quantum yield (0.25) and brightness compared with newer red proteins, so its readout is weaker and slower.
   • mKO2 For mKO2, two important issues are slow maturation and moderate acid sensitivity. FPbase lists a maturation time of about 108 min and a pKa of 5.5, so if the reaction is acidic or you measure too early, the orange signal may look weaker than the actual expression level.
   • mTurquoise2 mTurquoise2 is strong in cell-free systems because it has a very high quantum yield (0.93), which means it gives a bright signal even at moderate expression levels. It also has very low acid sensitivity (pKa 3.1) and a fairly fast maturation time of about 33.5 min, so its fluorescence is usually reliable across a wider pH range than many other FPs.
   • mScarlet_I mScarlet-I is useful because it was engineered for faster maturation than many older red fluorescent proteins; FPbase lists it at about 36 min. It is also very bright, but its moderate acid sensitivity (pKa 5.4) means the signal can still drop if the cell-free reaction becomes too acidic.
   • Electra2 Electra2 stands out because it is a bright blue fluorescent protein with FPbase brightness of about 61.48, which helps a lot when blue channels are usually dimmer. It also shows strong reported photostability in FPbase, so its signal should hold up better during repeated imaging of the same cell-free artwork.

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.

   • Hypothesis: To maximize mKO2 fluorescence over a 36-hour incubation, I would use the 2 µL reagent-addition window to add extra HEPES-KOH (pH 7.5) plus a concentrated GMP stock, or, if I had to stay strictly within the existing reagent list, extra guanine + ribose. I am targeting mKO2 because it has a relatively slow maturation time (~108 min) and moderate acid sensitivity (pKa ~5.5), so its final orange signal is especially vulnerable to both delayed chromophore development and pH drift during a long cell-free reaction.

   • Expected effect: Extra HEPES should improve buffering and keep the reaction closer to neutral over 36 hours, which should preserve fluorescence for an acid-sensitive orange FP like mKO2, while extra GMP should reduce the burden on the lysate’s purine-salvage pathway and help sustain the pool of GTP/NTPs needed for transcription later in the reaction. That matters because E. coli extracts can convert NMPs to NTPs in glucose-powered CFPS, but in your current mix the guanine side is starting from guanine with 0 GMP, so supplementing that branch should help maintain mRNA production for longer and ultimately increase total mature mKO2 fluorescence at the 36-hour endpoint.

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). **