Week 1 HW: Biomanufacturing with Xerophiles

Homework 1: Biomanufacturing Idea using Xerophiles / Extremophiles

(1) What I would like to biomanufacture and why

Target product: Deinoxanthin (and related carotenoid antioxidants) produced by the xerotolerant polyextremophile Deinococcus radiodurans.

Deinoxanthin is a unique carotenoid with strong antioxidant and radioprotective properties. It is more stable under oxidative stress and desiccation than many common carotenoids.

Why this is interesting:

• Deinococcus radiodurans naturally survives extreme dryness (xerotolerance) and high radiation.
• This makes it ideal for producing protective compounds for space missions, arid environments, or long-term storage of sensitive materials (vaccines, foods, cosmetics).
• The project connects sustainability, space biomanufacturing, and extremophile synthetic biology (inspired by Ye et al. 2023 and Averesch et al. 2023).

(2) What living system would I use?

Chassis: Deinococcus radiodurans

• Inputs: cheap carbon sources (sucrose or agricultural waste), minerals, moderate water.
• Process: Engineered carotenoid pathway (overexpression of key genes such as crtB and dxs) to increase deinoxanthin production. The organism already has native desiccation and radiation resistance.
• Outputs: Deinoxanthin-rich biomass that can be extracted.

Assumption: Recent genetic toolkits for Deinococcus make pathway engineering feasible.

(3) How would I use automation?

Primary tool for R&D: Opentrons OT-2 liquid-handling robot.

I would automate:

• Media preparation
• High-throughput strain screening
• Parallel small-scale cultivations
• Sampling and basic quantification of pigment

This speeds up the Design-Build-Test-Learn cycle. At larger scale, continuous or semi-open fermentation could be used, taking advantage of the extremophile’s robustness (less need for strict sterility).

(4) How much would I need to make, and how long would it take?

(a) How much?
Target: 10 kg of purified deinoxanthin (enough for a pilot space/medical application or small commercial batch).

Published titers are around 0.4 g/L. Assuming ~0.25 g/L recovered after processing → approximately 40,000 liters of culture.

(b) How long?
With optimized continuous or fed-batch fermentation in a few thousand liters of working volume, production of this amount could take several weeks to a couple of months. Lab-scale optimization with Opentrons would take weeks first.

(5) What don’t I know yet?

1. Actual yields under low water-activity conditions.
2. Robustness of current genetic tools for high-flux pathway engineering in Deinococcus.
3. Efficient and scalable downstream extraction and long-term stability of the purified product.

AI use disclosure
AI client: Grok (xAI).
I used it to research xerophiles/Deinococcus, structure the four required questions, estimate scale, and refine the proposal. All final scientific understanding and decisions are my own.

Week 2 HW: Genetic Circuit Design for Deinoxanthin

Week 2 Homework – Genetic Circuit Design

Part 0: Kernel Setup

Repository: awais-deinoxanthin
Folder: awais-deinoxanthin

Part 1: Expression Target

I designed a genetic construct to increase deinoxanthin production in Deinococcus radiodurans.

• Molecules: Two enzymes – CrtB (phytoene synthase) and Dxs
• Host: Deinococcus radiodurans
• Location: Cytoplasm

Part 2 & 3: Genetic Parts and Construct

I created the following parts in Kernel:

• Promoter: P_constitutive_Deinococcus
• RBS: RBS_strong
• CDS: crtB_Deinococcus
• RBS: RBS_strong_2
• CDS: dxs_Deinococcus
• Terminator: Terminator_strong

Construct name: Deinoxanthin_Expression_Cassette

Order of parts:
Promoter → RBS → crtB → RBS → dxs → Terminator

Part 4: Optimize and Quality Check

I codon-optimized the coding sequences for Deinococcus.
I checked the sequences using BLAST and ran the safety screen. No high-risk sequences were found.

Part 5: From Design to Build

For the first version, I will order the complete construct as synthetic DNA.
If multiple versions are needed later, I can use Gibson assembly.

Part 6: What I don’t know yet

1. Will the promoter work well in Deinococcus radiodurans?
2. Will the cells grow normally after overexpressing these two genes?
3. How much extra deinoxanthin will be produced?

AI Note
I used Grok to help structure the design and understand the Kernel workflow. The final decisions and understanding are my own.

Week 3 HW: Cell-Free Reaction Composition

Week 3 Homework – Designing Cell-Free Reaction Compositions

Overview

I designed cell-free protein synthesis (CFPS) reaction compositions for three proteins: sfGFP, PETase, and Reteplase using the coopetition.htgaa.org platform.

Compositions Submitted

1. sfGFP

• Type: Reducing condition
• Energy source: PEP-based
• Comments: Simple PEP-based composition based on reference plate.

2. PETase

• Type: Reducing condition
• Energy source: PEP-based
• Comments: Similar to sfGFP but suitable for an enzyme with one disulfide bond.

3. Reteplase

• Type: Oxidizing (DSB) condition
• Special reagents: GSSG, GSH, and DsbC
• Comments: Oxidizing composition to support formation of 9 disulfide bonds.

Community Reagent

I suggested Trehalose as a community reagent.
Reason: It acts as a protein stabilizer and can improve functional yield, especially for difficult proteins like Reteplase.

AI Note

I used Grok to help understand the requirements and structure the designs. Final decisions are my own.

Final Project