Week 3: Cell-Free Systems, Cloud Labs, & Field trip to Ginkgo Bioworks
HTBAA Homework 3 — CFPS REACTION DESIGN FOR SFGFP, PETASE, RETEPLASE
CFPS Arena → See how every coopetition design compares on $/g, explore the price map, and cost your own mix.
sfGFP (PDB 2B3P; UniProt P42212)
PETase (PDB 4CG1; UniProt Q6A0I4)
Reteplase (PDB 1RTF; tPA UniProt P00750)
Goal: using the collective intelligence of the HTBAA community and accessible AI systems, design novel CFPS reaction compositions for each of the three target proteins: sfGFP, PETase, and Reteplase to reduce the cost per gram of functional protein. Each design should be compatible with the experimental framework described above and should reflect the distinct expression and folding requirements of that protein. You can learn more about the details of the coopetition here.
A. COMPARE CELL-FREE REACTION COMPOSITIONS (20 minutes)
Why does the concentration of potassium glutamate and magnesium glutamate differ between compositions? Which is more sensitive?
Reasons for Differing Concentrations
The concentrations differ because these ions are essential for cell-free protein synthesis (CFPS) and the molecular interactions required for transcription and translation [5]. Specifically:
Optimization for specific strains: In some systems, the concentrations of these two ions are adjusted to account for the specific requirements of a given cell extract strain [5]. Mimicking the cytoplasmic environment: Research has shown that modifying salt concentrations to better recreate the E. coli cytoplasmic condition—such as using glutamate as the preferred anionic species—can influence protein expression [3]. Optimization goals: Different formulations aim for different outcomes, such as maximizing protein yield (e.g., the PANOx-SP system) or minimizing the reagent cost per gram of protein [1, 7]. Sensitivity
While the provided text does not explicitly state “magnesium is more sensitive than potassium” in a single direct sentence, it provides data regarding the sensitivity of different components:
Ammonium glutamate: Protein expression yields were noted to be “nearly insensitive” to ammonium glutamate concentrations between 0 and 100 mM [3]. Magnesium and Potassium Glutamate: One excerpt notes that the concentrations of these two ions are “essential” for CFPS [5]. In the context of optimization, researchers have performed sequential optimization of these ions (for example, optimizing magnesium glutamate between 1–6 mM while fixing potassium glutamate at a mean of 80 mM) to find the ideal balance for protein synthesis [5]. (Note: The specific comparison of which of the two—potassium or magnesium—is more sensitive is not explicitly answered in the provided excerpts, though they are both identified as key variables for optimization [5, 8].)
Sources consulted [1]p.10 HTBAA Homework #3 Cell Free Reaction Design Google Docs [2]p.5 [3]p.5 [4]p.4 Mimicking the Escherichia coli Cytoplasmic Environment Activates Long-Lived and Efficient Cell-Free Protein Synthesis (2004) [5]p.33 GENETICALLY EXPANDED CELL FREE PROTEIN SYNTHESIS SYSTEMS, METHODS AND KITS (2017) [6]p.16 [8]p.10 Using a GPT-5-driven autonomous lab to optimize the cost [7]p.2 Olsen2026 low cost reagents NatCommun
Explain how a PEP+NTP reaction formulation is different from a Ribose+NMP reaction formulation.
Based on the provided excerpts, there is no information that explains the difference between a “PEP+NTP” reaction formulation and a “Ribose+NMP” reaction formulation.
The excerpts do discuss different energy substrates and their effects on cell-free protein synthesis (CFPS), but they do not mention “Ribose+NMP” formulations:
PEP (Phosphoenolpyruvate): The text notes that PEP is a high-energy phosphate compound used to regenerate ATP [3]. However, using PEP as an energy substrate can lead to a rapid and sustained increase in phosphate levels, which can eventually inhibit protein synthesis [5, 7]. Pyruvate: The “Cytomim” system uses pyruvate as an energy source, which results in more stable pH and much lower phosphate accumulation compared to PEP-based systems [3, 5]. Other substrates: The text mentions “sacrificial substrates” like kinases and polyphosphate systems used to maximize yield, as well as the use of natural catabolic pathways like glycolysis and oxidative phosphorylation [8]. Because the term “Ribose+NMP” does not appear in the provided library, I cannot provide a comparison between the two specific formulations requested.
Sources consulted [1]p.10 [6]p.5 HTBAA Homework #3 Cell Free Reaction Design Google Docs [2]p.3 [3]p.7 [5]p.6 Mimicking the Escherichia coli Cytoplasmic Environment Activates Long-Lived and Efficient Cell-Free Protein Synthesis (2004) [4]p.5 [7]p.5 Olsen2026 low cost reagents NatCommun [8]p.9 Rasor2021 sustainable cell free COBIOT OSTI
Choose two cell-free reaction compositions from the literature, compare and contrast the choice of reagents and the concentration of reagents.
observatory Based on the provided literature, two cell-free reaction compositions that can be compared are the PANOx system and the Cytomim system [8].
Comparison of Reagents and Concentrations The primary differences between these two systems lie in their energy substrates, the types of glutamate used, and their buffering strategies.
- Energy Substrates and Metabolism
PANOx System: Utilizes Phosphoenolpyruvate (PEP) at a concentration of 33 mM as its energy substrate [8]. Cytomim System: Does not use PEP; instead, it uses Sodium pyruvate at a concentration of 33 mM [8]. 2. Glutamate and Salt Composition
PANOx System: Relies heavily on Potassium glutamate at a high concentration of 200 mM, but contains 0 mM of both Magnesium glutamate and Ammonium glutamate [8]. It also uses 80 mM Ammonium acetate [8]. Cytomim System: Uses a more balanced approach to glutamate. It contains 8 mM Magnesium glutamate and 10 mM Ammonium glutamate [8]. Its Potassium glutamate concentration (130 mM) is significantly lower than that of the PANOx system [8]. 3. Buffering and Other Additives
PANOx System: Uses HEPES KOH (57 mM) for pH buffering and includes Polyethylene-glycol (PEG) at 2% [8]. It also contains 2.7 mM Sodium oxalate [8]. Cytomim System: Uses Tris (2.4 mM) for buffering rather than HEPES [8]. It contains a slightly higher concentration of Sodium oxalate (4 mM) compared to the PANOx system [8]. Summary Table of Key Reagents | Reagent | PANOx System | Cytomim System |
| :— | :— | :— |
| Energy Substrate | 33 mM PEP [8] | 33 mM Sodium pyruvate [8] |
| Potassium glutamate | 200 mM [8] | 130 mM [8] |
| Magnesium glutamate | 0 mM [8] | 8 mM [8] |
| Ammonium glutamate | 0 mM [8] | 10 mM [8] |
| Buffering Agent | 57 mM HEPES KOH [8] | 2.4 mM Tris [8] |
Sources consulted [1]p.10 [3]p.10 [4]p.2 HTBAA Homework #3 Cell Free Reaction Design Google Docs [2]p.33 GENETICALLY EXPANDED CELL FREE PROTEIN SYNTHESIS SYSTEMS, METHODS AND KITS (2017) [5]p.1 [6]p.10 [7]p.2 Olsen2026 low cost reagents NatCommun [8]p.2 Mimicking the Escherichia coli Cytoplasmic Environment Activates Long-Lived and Efficient Cell-Free Protein Synthesis (2004)
Note: A recommended starting point would be to review a least one published formulation, such as PANOx-SP (Jewett & Swartz 2004), a cost-optimized formulation from Olsen et al. (2026), and/or an AI-designed formulation from Smith et al. (2026).
B. DESIGN ONE REACTION COMPOSITION FOR EACH OF THE THREE PROTEINS (40 minutes)
Sign in to coopetition.htgaa.org and publish your first unique 20 µL CFPS reaction composition for each of the following (don’t worry, you can resubmit later): sfGFP PETase Reteplase
Note: if you’re unable to login with your HTGAA keycloak account, please post in Zulip so that you can participate in the coopetition!
For each composition, specify the reagents you would adjust from the relevant categories, including salts, energy sources, building blocks, cofactors, and redox components.
As a HTBAA research team, we will have the opportunity to suggest new reagents to add to the cell-free reagent inventory. Contribute (or upvote) at least one reagent to the “Community Reagents” section of the website and provide a short rationale. Include in your homework documentation how you decided on that specific reagent and provide links to any relevant research.
Visit the cfps-coopetition forum page to ensure you will be able to take part in the discussion. Bonus: send a note in the channel about a CFPS optimization idea that you have! If you would like clarification on aspects of the project, please message in this dedicated channel.
C. ASIMOV KERNAL (OPTIONAL)
Build T7 compatible plasmids for cell-free expression using the amino acid coding sequences below:
Bonus question: design a T7 helper plasmid that you could add to the cell-free reaction to improve the yield of functional reteplase (general chaperones, disulfide-bond machinery, etc).
sfGFP MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEKGTRDHMVLHEYVNAAGITWSHPQFEK
PETase MEKKIWSHPQFEKGGSGENLYFQSGGASPAGGGANPYERGPNPTDALLEASSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGTEASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHSMGGGGTLRLASQRPDLKAAIPLTPWHLNKNWSSVTVPTLIIGADLDTIAPVATHAKPFYNSLPSSISKAYLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPFGGSGVSGWRLFKKIS
Reteplase MEKKIWSHPQFEKGGSGENLYFQSGGASPAGGGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRPGGSGVSGWRLFKKIS