Week 1 HW: Final Project Ideas

(1) What you would like to biomanufacture and why?

Describe the target product or output you would like to make.

Why are you interested in making it? Your motivation might be utilitarian—for example, addressing a problem in health, sustainability, food, energy, or manufacturing—but it does not need to be. Your project might instead have a social, cultural, artistic, or personal motivation. Clearly articulate what you want to make, who or what it is for, and why you think it is interesting or important.

I would like to make glow-in-the-dark ink using bioluminescent microbes. This is partially personally motivated, as I am an avid reader and would like to not have to find a good light source all the time, but it is also motivated by sustainability, as this could be used to make road signs or markings that glow at night and allow drivers, pedestrians, etc. to see better without using electricity.

(2) What living system would you use?

Propose at least one living-system manufacturing chassis that could produce your target—for example, a microbial, mammalian, or cell-free system.

At a high level, describe how the system would make your target product. What goes into the system? What biological process or processes occur? What comes out?

You do not need to design the complete biological system yet. Make reasonable assumptions where necessary and identify them.

I would use bacteria, perhaps a species that already has bioluminescent properties and engineer it to reliably glow at night/when there is a lack of light. This way, during the day, the microbes are also visible but not burdened by producing light. Bacteria are also helpful as they can form biofilms and persist better that way, which would be good for road markings since they are out in the elements.

(3) How would you use automation?

Identify at least one automation technology that you would combine with your living system.

This could include a large-scale Cloud Lab, a desktop liquid-handling robot such as an Opentrons, a programmable microfluidics platform such as Nuclera’s, or another automation technology.

Describe what part of the biomanufacturing workflow you would automate and why. For example, you might automate liquid handling, culturing, assembly, screening, sampling, measurement, optimization, or another step.

I would like to automate the assembly of the gene circuit I would be using to ensure the bacteria reliably glows below a certain light level and to encourage biofilm formation. Some papers have suggested that the Opentrons robot is capable of Golden Gate assembly (Bryant et al), so I could use that for this. It would also be helpful to automate culturing of the bacteria, as this would have to be made in large quantities if it is to be used widely. I would also use a Cloud Lab so that the circuit made by the Opentrons can automatically be fed into equipment that would electroporate the construct into the bacteria, screen for transformants, and grow it up.

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

Perform a back-of-the-envelope calculation estimating what it would take to manufacture your target at a meaningful scale.

(a) How much?

Define a scale relevant to your project and calculate approximately how much of your target product you would need to manufacture.

For example, if you are producing a medicine, you might estimate the amount required to treat 1,000 people, everyone in Massachusetts, or everyone in the world. If you are producing a building material, you might calculate the amount required for one structure or an entire neighborhood. If you are producing an art or design artifact, define an appropriate scale yourself.

Clearly show your calculation, including units, assumptions, and sources for important numbers.

For Williamsburg, there are ~50 miles of road (Street Division).

According to this site: https://www.cojoasphalt.com/blog/traffic-paint-coverage-rate-calculator, a 4-inch stripe at 15 mil converts to 320 ft/gal paint.

Assuming a two-way road, there are four stripes (two white solid lines at the edges of the road, and the double yellow stripe down the middle) Ignore other markings like crosswalks, lane arrows, etc. for simplicity.

Total volume of paint needed: $50 \text{ miles} = \frac{5280 \text{ ft}}{1 \text{ mile}} * \frac{1 \text{ gal}}{320 \text{ ft}} = 825 \text{ gal}$

(b) How long?

Using your proposed living system and automation technology, estimate how long it would take to manufacture that amount.

Consider quantities such as production rate, yield, volume, number of parallel experiments or reactors, automation throughput, or whatever parameters are most appropriate for your system.

You may discover that the automation technology you would use to prototype your system is very different from what you would use to manufacture at scale. If so, describe both.

Again, we are looking for a reasonable engineering estimate. Show your assumptions and calculations.

To prototype my system, I would manually construct the circuit using a Thermal Cycler and a 100-250 mL culture in a shaking incubator.

For producing at scale, let’s assume I’m using Vibrio fischeri, the bioluminescent symbiont in Hawaiian bobtail squid that glows at night using quorum sensing (Nyholm et al).

Creation of electrocompetent cells following a protocol by Gonzales et al: including the step for growing up an overnight culture, 20 hours.

Assembly of construct: let’s assume I’m using Golden Gate assembly. According to NEB, the protocol is 30-60 5 minute cycles, but I want to make large quantities of construct, so we’ll choose 60 cycles. This ends up being a 5 hour reaction.

Electroporation does not take long, so we’ll say that’s one hour.

Then, plate the bacteria and allow them to form colonies on selective media to screen for transformants (the construct would have an antibiotic resistance gene, and the media would have the antibiotic). The plate would incubate for a day (24 hrs).

Then, select a colony to start a culture from, which takes 1-2 hours to grow to log phase (Christensen et al).

In total, this is 52 hours (a little over 2 days). However, the time to grow up the final culture would likely take longer, as I would be growing the bacteria in larger volumes but would want to inoculate still with a small volume to conserve energy/resources.

5. What don’t you know yet?

After completing your analysis, identify the 2–3 biggest technical unknowns, bottlenecks, or assumptions in your proposed biomanufacturing system.

What would you need to learn, measure, or experimentally determine to know whether your idea could actually work?

1. Whether it’s possible to engineer a system that responds to the lack of light. I’d experiment with light-sensitive proteins that change conformation in the presence of light to inactivate the bioluminescent protein.

2. Whether my chassis (whether its V. fischeri or not) would be able to survive on pavement/on paper. I would have to research types of bacteria that would grow in these conditions and their propensity for forming biofilms.