Week 11 Homework

Week 11 Homework: Bioproduction and Cloud Labs

Part A: The 1,536 Pixel Artwork Canvas

I contributed two pixels on the bottom left of the artwork, but for some reason they did not sync or show up correctly in the final version. I still liked the idea of the project because it turned a biology class assignment into a shared artwork, where everyone’s tiny contribution could become part of a larger image.

Part B: Cell-Free Protein Synthesis

1. Roles of each component

E. coli lysate: Provides the cell machinery needed for transcription and translation, including ribosomes, enzymes, tRNAs, and factors. The BL21(DE3) Star lysate also includes T7 RNA polymerase.

Potassium glutamate: Helps set the salt conditions for the reaction. Potassium is important for translation.

HEPES-KOH pH 7.5: Buffers the reaction so the pH stays near 7.5.

Magnesium glutamate: Provides magnesium, which is needed for ribosome function and nucleic acid stability.

Potassium phosphate monobasic and dibasic: Help buffer the reaction and provide phosphate.

Ribose: Helps support nucleotide regeneration in the longer reaction.

Glucose: Provides a longer-lasting energy source through metabolism in the lysate.

AMP, CMP, GMP, and UMP: Nucleotide monophosphates that can be converted into forms needed for RNA synthesis.

Guanine: Supports guanine nucleotide salvage and helps rebuild GTP.

17 amino acid mix: Provides most of the amino acids needed to make protein.

Tyrosine and cysteine: Added separately because they are less stable or need separate handling.

Nicotinamide: Supports NAD-related metabolism in the reaction.

Nuclease-free water: Brings the reaction to the final volume without degrading DNA or RNA.

2. Difference between the 1-hour and 20-hour master mixes

The 1-hour PEP-NTP mix is a faster, more direct system because it provides ready-to-use NTPs and a high-energy phosphate source. The 20-hour NMP-ribose-glucose mix is designed to last longer by using monophosphates, ribose, glucose, and lysate metabolism to regenerate energy and nucleotides over time.

3. Bonus

Transcription can still happen if guanine is included because the lysate can convert guanine through salvage pathways into GMP, then into GDP and GTP. So GMP does not always need to be added directly.

Part C: Planning the Global Experiment

1. Fluorescent protein properties

sfGFP folds well and is usually reliable in cell-free systems, so it should give a strong green signal.

mRFP1 is a red fluorescent protein, but it can mature more slowly and may not be as bright as newer red proteins.

mKO2 is an orange fluorescent protein. Its signal depends on proper folding and chromophore maturation.

mTurquoise2 is a cyan fluorescent protein with relatively strong brightness, but it still depends on folding and oxygen for maturation.

mScarlet-I is a bright red fluorescent protein. Its final signal may depend on giving it enough time to fold and mature.

Electra2 has a different excitation and emission profile, so the readout depends on matching the plate reader settings well.

2. Hypothesis

I would focus on mScarlet-I. My hypothesis is that using the longer NMP-ribose-glucose energy system and slightly increasing magnesium glutamate would improve red fluorescence after 36 hours. The longer energy system should keep protein production going, and magnesium should help translation. The expected result is a stronger final red signal.