Week 11 HW: Building-genomes

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

  1. Contribute at least one pixel to this global artwork experiment before the editing ends on Sunday 4/19 at 11:59 PM EST. A personalized URL was sent to the email address associated with your Discourse account, and you can discuss the artwork on the Discourse. If you did not have a chance to contribute, it’s okay, just make sure you become a TA this fall! 😉

  2. Make a note on your HTGAA webpages including: what you contributed to the community bioart project (e.g., “I made part of the DNA on the bottom right plate”) what you liked about the project, and what about this collaborative art experiment could be made better for next year.

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

BL21 (DE3) Star Lysate (includes T7 RNA Polymerase) Salts/Buffer

Potassium Glutamate HEPES-KOH pH 7.5 Magnesium Glutamate Potassium phosphate monobasic Potassium phosphate dibasic Energy / Nucleotide System

Ribose Glucose AMP CMP GMP UMP Guanine Translation Mix (Amino Acids)

17 Amino Acid Mix Tyrosine Cysteine Additives

Nicotinamide Backfill

Nuclease Free Water 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)

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

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

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.

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.

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.

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