Week 11 HW: Bioproduction & Cloud Labs

Part A: The 1,536 Pixel Artwork Canvas | Collective Artwork

I couldn´t contribuite to the pixel artwork sadly :(

Next year i become a TA without excuses.

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

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.

E. coli Lysate: Provides the cellular machinery for transcription and translation, including ribosomes, tRNAs, enzymes, and metabolic proteins.

BL21 (DE3) Star Lysate: A lysate strain optimized for protein expression. It includes T7 RNA polymerase, which transcribes genes controlled by a T7 promoter.

Potassium Glutamate: Maintains ionic strength and mimics the intracellular salt environment needed for efficient translation.

HEPES-KOH pH 7.5: Buffers the reaction to keep the pH stable near physiological conditions.

Magnesium Glutamate: Provides Mg²⁺, essential for ribosome function, RNA stability, and enzyme activity.

Potassium phosphate monobasic: Helps buffer the reaction and contributes phosphate for energy metabolism.

Potassium phosphate dibasic: Works with monobasic phosphate to maintain pH and phosphate balance.

Ribose: Carbon precursor used to regenerate nucleotides and sustain long-term transcription.

Glucose: Energy source that feeds metabolism in the lysate to support ATP regeneration.

AMP: Precursor for ATP and RNA synthesis.

CMP: Precursor for CTP and RNA synthesis.

GMP: Precursor for GTP and RNA synthesis.

UMP: Precursor for UTP and RNA synthesis.

Guanine: Purine base that can be salvaged by lysate enzymes to make GMP/GTP.

17 Amino Acid Mix: Supplies most amino acids needed for protein translation.

Tyrosine: Added separately because it has lower solubility or different preparation needs.

Cysteine: Added separately because it is chemically sensitive and can oxidize.

Nicotinamide: Supports NAD-related metabolism and helps sustain long-duration energy regeneration.

Nuclease Free Water: Used to bring the reaction to the final volume without adding nucleases that could degrade DNA or RNA.

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.

The 1-hour PEP-NTP mix uses ready-made NTPs and PEP for fast, immediate transcription and energy supply. The 20-hour NMP-Ribose-Glucose mix uses NMPs, ribose, glucose, phosphate, and guanine so the lysate can regenerate nucleotides and energy more slowly and sustainably over long incubation. It is designed for longer protein production rather than maximum short-term speed.

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

Guanine can be converted by salvage-pathway enzymes in the E. coli lysate into GMP, then further phosphorylated to GDP and GTP. That regenerated GTP can then be used by T7 RNA polymerase during transcription.

Part C: Planning the Global Experiment | Cell-Free Master Mix Design

Given the 6 fluorescent proteins, identify and explain at least one biophysical or functional property of each protein that affects expression or readout in cell-free systems.

sfGFP: Folds very efficiently and matures well, so it usually gives strong fluorescence in cell-free reactions.

mRFP1: Often matures more slowly and can be less bright than newer red fluorescent proteins, reducing signal during shorter incubations.

mKO2: Its orange fluorescence depends on proper folding and chromophore maturation, which can be affected by oxygen and reaction conditions.

mTurquoise2: A cyan fluorescent protein with good brightness, but readout depends strongly on correct excitation/emission settings and folding.

mScarlet_I: Very bright red fluorescent protein, but full fluorescence still requires proper folding and oxygen-dependent chromophore maturation.

Electra2: Fluorescence output depends on efficient expression, folding, and maturation; its signal may be affected by pH, oxygen, and reaction duration.

Create a hypothesis for how adjusting one or more reagents in the cell-free mastermix could improve fluorescence over a 36-hour incubation.

For mScarlet_I, increasing glucose/ribose-based energy regeneration and maintaining HEPES/phosphate buffering should improve long-term translation and chromophore maturation over 36 hours. Expected effect: more properly folded and matured red fluorescent protein, giving higher final fluorescence.

For the following parts i sadly wasnt`t able to continue because i couldn´t send the master mix.