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Two PDF's are available, the detailed proposal description of the project and the short proposal description.
Summary;
Play is an important part of culture and, therefore, society. Play lets us learn, connect, and create. Unfortunately, play is not so well integrated into biotechnology. The main reason? Not everybody can play with biotechnology. Because to do so, you need special and expensive equipment and a particular skill set. In other words, playing with biotechnology is a privilege. The lack of play with biotechnology means that there are too few connections between society and biotechnology. This causes misunderstandings about the field, like the misconception of vaccines or the reluctancy to accept GMOs. This is a shame because both vaccines and GMOs have the potential to help the world become a better place. Project N.I.N.O. makes it possible for anyone to play with biotechnology.
N.I.N.O. stands for Nifty Incubator for Nurturing Organisms. It is a pocket-sized bioreactor containing micro-organisms based on the pulse-fed-batch principle. You can look at it like a biological Tamagotchi. Just like the 90s Tamagochis, you will need to feed your micro-organisms to keep them alive. The micro-organisms will also give you feedback on how you are doing. If they are stressed, they will produce a red pigment, and when they are growing they will inflate flagella.
The objective of N.I.N.O. is to connect people through biotechnology, connect people to biotechnology, and showcase the cool things you can do with biotechnology. This is all achieved by providing a microbial playground that anyone can use.
The project consists of a couple of phases. First, a micro-organism is selected and a specialized strain is constructed that is able to communicate by producing a red pigment. Next, this strain is characterized and the most important parameters are obtained. These parameters are then used to come up with a biochemical design of the N.I.N.O. This biochemical design is made with the growth calculator. When this is set, a physical design can be made.
The deliverables consist of a working prototype, a presentation of this prototype, a blueprint (protocols) on how to make the N.I.N.O., and a N.I.N.O. suitable strain.
The working prototype needs to be turned into version one in order to achieve the stated objectives. If the project is deemed a success, this version 1 could be produced and distributed. An example of an event that suits the N.I.N.O. very well is the IGEM competition 2023.
All in all, Project N.I.N.O. has the potential to create a playful interface between biotechnology and society. It is therefore a perfect fit for the Ginkgo creative residency of 2022.
Introduction;
Play brings happiness and pleasure, it brings people together, play creates culture, it is an intrinsically motivated activity, play has rules but leaves room for creativity and play is imaginative. In conclusion, play is one of the most important elements of life! It are the moments of play in your work that generate the best ideas and solutions to problems.
This was also seen in the discovery of the structure of DNA, where Watson and Crick used all the available information known that day and laid the final puzzle pieces by playing with a model of the structure of DNA. The other way around play is used to aid scientists with their work, a good example is fold.it. By playing with the protein structures gamers are helping scientist design software to predict protein structures1.
Play is often used to demonstrate some of the most cutting-edge techniques. Consider the work of Fernandez and Voigt2. They show off their genetically modified E. coli that can generate images. At first glance these pictures don’t serve any function, they have a very bad resolution, are nearly impossible to conserve, and take very long exposure times. They were essentially playing with their technique of gene switches. This gene switch system could be used for a lot of application in medicine or microbial production that bring a lot of value to this world.
Finally, playing can also spark interest into subjects. A perfect example of this is Charles Babbage, when he was young he was invited to Merlin’s mechanical museum where he played with the mechanical automatons from inventor John-Joseph Merlin. Because of this encounter he became fascinated with automatons and eventually invented the frontrunner of the programmable computer3.
In biotechnology, play has long been a privilege. You need many resources, like a lab, expensive consumables, and some knowledge. With the stunning decrease of cost and increased efficiency of the techniques in the past 2 decades this privilege is contested. However, a breakthrough is required. A break through that Project N.I.N.O. may be able to force.
This document describes project N.I.N.O. which stands for Nifty Incubator for Nurturing Organisms. N.I.N.O. is a vehicle for a population of micro organisms, which the player can nurture in a similar way as the 90’s tamagochi or a SIMS character. N.I.N.O. has the potential to connect people through biotechnology, by providing a community where people can discuss how they play and share knowledge. N.I.N.O. connects people to Biotechnology, by nurturing micro-organisms you start to feel a connection between you and your population. N.I.N.O. can showcase the potential of biotechnology by using genetically modified organisms and transparently discussing how they were constructed.
The structure of this proposal is as follows. First the relevancy of the project for the biotech field is discussed. It answers the question on why it is important to play with biotechnology and how project N.I.N.O. is accomplishing this. Next, the scope of the project is specified. It shows the project goals and deliverables. It also has a paragraph describing the ideal outcome of the project. Then, we have the project description which consists of two parts, the N.I.N.O. design and the biochemical design. The N.I.N.O. design goes into the physical part of the N.I.N.O. It lists its requirements and functional capabilities. The biochemical design describes a method of how the micro organism can be transformed to give feedback to its user. It also describes the growth calculator which is used to successfully design the biochemical important parameters of the design. After the project description the next chapter is the project planning consisting out a timeline, important milestones, and an estimation of the costs. Finally, the future of N.I.N.O. after the project is discussed and how it could be used to let a big audience play with biotechnology.
Relevancy of the project;
As explained in the introduction, play creates culture. Culture is a big part of any society. Hence, play is an important part of society as well. Unfortunately, it is difficult to play with biotechnology. For many people, the only time they are in direct contact/or playing with biotechnology is when they are baking bread, brewing beer, or making kimchi. Most of the biotechnology field is only playable by a fortunate group of people, like scientists and students. The need for a laboratory, the high cost of consumables, and a necessary skill set are hurdles that not everybody can overcome.
With the N.I.N.O. all these hurdles are overcome; it is cheap, it has no lab requirements, and everybody is able to use it. When more people play with biotechnology, biotechnology will be a bigger part of society, which is important considering the potential of this field and the aversion some people have to it.
SCope;
In the paragraph below, an overview of the scope is shown. It shows what is in scope and what is out of scope. Underneath, a more detailed explanation is given why certain attributes are in or out of scope.
In scope
- The physical design of N.I.N.O
- Micro-organism design
- Biochemical design
- I.N.O community design
- A working prototype of the N.I.N.O
Out of scope
- Multiple designs
- Fabrication of numerous N.I.N.O V1
A physical design is needed to end up with a working prototype. For more information on the requirements of this design, read this paragraph. It is necessary that the micro-organism inside the N.I.N.O can communicate with the player. Without such communication, it will be difficult to grasp how the organism is feeling. That is why the micro-organism needs to be designed to be able to communicate in order to achieve a working prototype. Read this chapter for a more detailed description of how this is accomplished. A biochemical design is also needed for the working prototype. Before building the N.I.N.O., you need to know the volumes of the different reservoirs and their flows. These should be designed in such a way that the game is playable. For more information, see this section. One of the objectives is to connect people through biotechnology. That’s why a blueprint will be made on how you can best set up a N.I.N.O. community. More information on this topic can be found here. The end goal is, of course, also part of the scope. The working prototype is the apotheosis of the project.
It is easy to make different designs of the N.I.N.O. For example, with slower growing organisms to elongate the play time you have with one N.I.N.O. However, because of the limited time of the project (3 months), it is important to keep focus. Therefore, alternative designs are out of scope. While it would be very cool to have large quantities of N.I.N.O.s at the end of the project, it is again the limited amount of time that excludes it from the project.
Deliverables
Project N.I.N.O. has the following deliverables.
- Working prototype N.I.N.O.
- Transformed and characterized micro-organism that works with N.I.N.O.
- Presentation and demonstration of Project N.I.N.O.
- Protocols on how to design the N.I.N.O. and its organism.
The ideal outcome
If this project is chosen, the ideal outcome would be a working prototype. This working prototype could afterwards be changed into version one. This version meets all the requirements for distribution. The N.I.N.O. could be handed out in schools and conferences, or sold online to individuals at the cost price.
The Design;
This section consists of N.I.N.O.’s physical design and the biochemical design. The physical design lists the design requirements and physical components of the N.I.N.O. The biochemical design shows the design proposal of the micro-organism used in N.I.N.O. and a growth calculator that is able to model the reactor. This is important to obtain the necessary concentration, volumes, and feeds of the reactor.
The physical design
The physical design of N.I.N.O. depends on the design requirements listed below. These requirements ensure that N.I.N.O. can be used as intended.
Design requirements
- Costs less than 10 dollars to produce
- Pocket size
- Durable (no leakage)
- Playable for 5 days
- Feedback from micro-organism
- Easy to operate
- Of-the-shelf parts
- Open source
- Symmetrical aesthetics
The N.I.N.O. production cost cannot be higher than 10 dollars. If the cost are too high a smaller group of people can afford N.I.N.O. This would undermine the objective of letting anyone play with biotechnology. The N.I.N.O. should be pocket size. Because the microorganisms are your babies, you must be able to transport them wherever you go! I finally understand why some people prefer chihuahuas over normal sized dogs. The design should be durable in order to protect both the player and the microorganisms. Meaning that no leakage can occur when used in normal circumstances. The aim is to make the game playable for 5 days. This could change if the characterization yields unexpected results. The user should be able to receive feedback on its performance. In other words, the micro-organism should be able to show whether it is happy or not happy by communicating with the player. Using the N.I.N.O. should be easy. It should be as easy as operating a water ring toss toy. The N.I.N.O.'s ease of use results in a larger audience that can be reached. The N.I.N.O. should be built with off-the-shelf parts. This makes it cheaper to produce and it makes it easier for the community to adapt N.I.N.O. to their liking. This is also why the blueprints of N.I.N.O.'s design should be open source. Finally, the aesthetics should be symmetrical, meaning the design has no indication of how to use the device.
Design schematic
Figure 1 shows a schematic of the N.I.N.O’s design. The bioreactor is the device's main component. The microorganisms are hosted in this part, which is visible to the player. The maximum volume inside the bioreactor is greater than the initial volume. This is because the design of N.I.N.O. makes it a fed-batch system. To prevent anything growing, the initial volume only contains phosphate buffer. This is even true when the start button is pressed. The biochemical design determines the initial volume and bioreactor volume. Next to the bioreactor there are 2 more reservoirs containing different solutions. The first is the feedstock reservoir. The water-based solution inside contains a minimal medium with glucose as the carbon source, ammonia (NH4+) as an N-source, and vitamins. The second reservoir contains the inoculum (the micro-organisms). Adding the biomass to the bioreactor starts the N.I.N.O. For every reservoir, a pump is installed to pump the liquid from the reservoir to the bioreactor. These pumps pump a set volume per activation. The pumps are activated by pressing the pump. The force of pressing down will push the liquid through its tubing. No electricity is used in the device. All the energy comes from kinetic energy produced by the user. The micro-organism will produce gas (CO2). This gas can be used to communicate with the player. It can be caught in a (growing) balloon or syringe. It can be designed to be the finish line. For example, if the balloon bursts, the game is finished or it can be used to transform the device (f.e., grow flagella).
Figure 1: The schematic of the N.I.N.O.
The biochemical design
The Micro-organism
The micro-organism is the heart of the project. It is what makes N.I.N.O playable. N.I.N.O. can be used with any type of microorganism. However, it is important for the micro-organism to give feedback to the user when it is not feeling well. Microorganisms can communicate with humans in a variety of ways, such as through smell or color. For this project, it was chosen to use color as a medium for communication. The following section explains how communication via color could be introduced into a micro-organism.
Using only one method of communication keeps the design simple. As a result, it is determined the micro-organism will only communicate when it is stressed. Stresses arise from high osmotic pressure, low or high pH, starvation due to a lack of nutrients, mechanical friction, and temperature. It would be difficult to engineer a sensor for all these different forms of stress. Therefore, a general stress marker called RpoS is used to produce a pigment6. This is done by using a RpoS sensitive promotor that upregulates the expression of a red fluorescent protein (RFP)5. RpoS reaches a very high concentration when the bacteria faces stress. The RpoS will change the expression of many genes to make the bacteria more resilient to stress7. It is important to note that not all bacteria have this RpoS system, especially some laboratory strains that have lost their RpoS. The loss of RpoS can be advantageous because switching the gene expressions takes an investment of energy8. It is therefore important to check if the strain used is not RpoS impaired.
Figure 2 shows the design of the suggested transformation into the bacteria. The transformation is relatively simple since only one gene is introduced. The first part of the insertion consists of a promotor (fig. 2A). This promotor should be upregulated by RpoS. Pdps is the promotor of the dps gene in bacteria and is known to be upregulated by the presence of RpoS8. The second gene is a red pigment. A good option is using a red fluorescent protein(RFP). There are many variants of RFPs. Suggested are mCherry, mStrawberry, and mPlum9. Finally, a terminator is needed. The exact method of the construction and transformation of this gene depends on the parts library and method used at Ginkgo.
Figure 2: The design of the inserted gene in the N.I.N.O. micro-organism.
Growth calculator
The playability of the game depends on the type of micro-organism used and the concentrations of substrate, products, and biomass over time. As well as the volumes of the different reservoirs and feeds. A growth calculator is made to calculate start concentrations and volumes. The growth calculator has a series of inputs. These inputs can be categorized into inputs depending on the micro-organism used, starting concentrations, and the N.I.N.O. design. The growth calculator is available via this link.
Micro-organism dependent inputs
The Yield (Yx/s)
The yield is the ratio between the amount of biomass produced over the amount of substrate consumed in kgX/kgS. The value is between 0 and 1, and the higher the value, the more efficient the micro-organism is using the available substrate for growth. This is a value that depends on the micro-organism and should be calculated with an experiment.
The maximum growth rate (µmax)
The maximum growth rate is the maximum growth the micro-organism achieves during exponential growth (no nutrient limitation in ideal circumstances). The higher this value, the shorter the doubling time of the micro-organism is. The value depends on the micro-organism and should be calculated with an experiment.
The substrate specificity constant (KS)
The substrate specifity constant is the substrate concentration where the growth rate is exactly half of the µmax. This is a micro-organism specific constant and should be obtained from experiments. The units of the constant is kgS/m3.
The cell specific death rate (Kd)
While organisms have a cell specific growth rate (µ), they also have a cell specific death rate. When the cell specific growth and death rate are equal, the organism enters the stationary phase.
The product yield constant acetate(Yp,acetate/s)
In anaerobic fermentations, micro-organisms can not fully combust their substrates. Meaning that there are some catabolic products. While in general, products are not interesting for N.I.N.O., some products have an inhibitory effect on the growth rate depending on their concentration. Acetic acid/acetate is a product that is known to have an inhibitory effect on the growth rate10,11.
The product yield constant ethanol(Yp,ethanol/s)
Another product of the fermentation that has an inhibitory effect is ethanol15. The yield is necessary to know to calculate the effect it has on the growth rate.
The product specific inhibitory constant acetate (KP,ace)
Acetate has an inhibitory effect on the growth of micro-organisms. The specific inhibitory constant is necessary to calculate this effect.
The product specific inhibitory constant ethanol (KP,eth)
Ethanol has an inhibitory effect on the growth of micro-organisms. The specific inhibitory constant is necessary to calculate this effect.
The product yield constant CO2(YP,CO2/S)
The volume of gas produced during the fermentation is an important design parameter. The size of the off gas container depends on it. To calculate the volume of gas produced, you need the yield of CO2 over substrate.
Starting concentrations
Substrate concentration at t=0 (CS0)
The starting concentration of substrate is important for the growth curve and rate of the organism. If the concentration is very high, the organism can grow at its maximum growth rate for a longer time and additional feeding is not necessary. If it is zero, the micro-organisms will not grow unless feed containing substrate is added.
Substrate concentration in the feed (Cs,in)
The substrate concentration in the feed together with the feed volume determines the amount of substrate entering the reactor when the feed button is activated.
Biomass concentration at t=0 (CX0)
The initial biomass concentration is important, because a high concentration will cause the game to end earlier. This amount is therefore dependent on the designed tfinal (max total time of the game).
Starting volume in the bioreactor (V0)
The starting volume of the bioreactor is important because it determines the left-over volume to be fed into the reactor.
Activation interval (h)
The activation interval is the time between two activations of the feed button. The shorter the interval the more the reactor is fed. Feeding every 10 hours might be a bit boring, while feeding every 5 minutes is too much.
N.I.N.O. design dependent inputs
The maximum bioreactor volume (Vmax)
The maximum volume of the bioreactor is the limited factor of the fed-batch principle of the N.I.N.O. bioreactor.
Feed volume per activation (Fact)
The amount of added volume when a button/pump is activated.
Calculations performed by the model
These inputs are used to calculate the further course of the reactor. With this information, you can design the N.I.N.O in a way that it is playable. The most important parameters are the concentration of biomass, the concentration of substrate (glucose), the growth rate, the concentration of ethanol, and the pH.
Euler steps model explanation
The calculations described below consist mainly of derivatives. The easiest way to solve these derivatives is to use the Euler’s method or Euler steps. It is a numerical method to approximate the actual outcome. The Euler method takes small time steps and evaluates the direction of the point in the graph. In this case, the X-axis is the time and the Y-axis the concentration of a certain compound. Subsequently, the direction is the rate of change of concentration of this compound. When you take this direction/slope at a time point X, you can approximate the Y value at X+1. The smaller steps you take, the more accurate the model calculates. In this case, we use a step of 1 second. On a whole day (86400 seconds), these steps are quite small. Therefore, the approximation is also quite good.
The growth rate (µ) is dependent on the maximum growth rate (µmax), the concentration of substrate (CS and KS, blue part of the equation), the concentration of acetate (CP,ace and KP,ace, red part of the equation), the concentration of ethanol (CP,eth and KP,eth, pink part of the equation). The formula is depicted below this paragraph. The growth rate is important for project N.I.N.O. because a low growth rate for a longer period of time will cause the bacteria to produce the red dye. If the calculations show that with the design of the bioreactor it is impossible to keep the µ > 0, the design should be altered because the game is deemed unplayable.
The actual rate of change in biomass due to growth depends not only on the growth rate but also the concentration of biomass available at that point in time (CX). This rate is later used to determine the amount of biomass at a certain time point.
Next to the growth rate of the bacteria, there is also a death rate (Kd) that is always there. The actual change of biomass due to this deathrate depends also on the available biomass at a certain time point (CX).
Subsequently, the actual rate at which biomass changes (rX) is the change due to growth minus the change due to death.
The change in substrate (rS) is calculate using the yield of biomass on substrate (YX/S) and the rate of biomass growth (rX,growth), since the energy of the substrate is used to grow biomass.
Because the growth is anaerobic, the sugar can not be combusted, but is changed into a variety of other catabolic products. Two of these products, acetic acid and ethanol, are interesting because they have an inhibitory effect on the growth of the micro-organism. The next two equation show the calculation of the rate, which is based on the yield of acetate on the substrate and likewise for ethanol.
Another catabolic product of interest is CO2. This is a gas and its volume is important to know because it determines the size of the off gass reservoir. Because it is a catabolic product the rate of production is depend on the substrate rate and the yield of CO2 over the substrate.
Now that all rates are defined it is possible to calculate the mass balances over time for the biomass, the substrate, and three two products. First the substrate, the amount of substrate over time in the bioreactor is depending on two parts, the amount of substrate entering the reactor at that moment and the consumption of the substrate already in the reactor. This yields the following equation:
Next up is the biomass, this mass balance also consists of the two parts, the increase of biomass and the income concentration of biomass. Since, the inflow does not contain any biomass a flow pulse decreases the concentration of biomass.
The mass balances of acetate and ethanol are produced in a similar way because they as well increase over time.
Because CO2 is a gas and the assumption is made that it is not dissolving in the solution. The mass balance is equal to the rate of CO2 production.
To calculate the amount of off gas produced Henry’s law is used. Henry’s law states that the concentration of a gas in a solution is proportional to the partial pressure of this gas and the concentration in the liquid. This proportion is summarized by the henry’s constant (KH).
The KH of CO2 is 0.037 mol/L*atmosphere. It is assumed that only CO2 is present and that the N.I.N.O. is under atmospheric pressure. This means that the partial pressure is 1 atmosphere. This means that the concentration of CO2 in the bioreactor needs to be >0.61 g/l before off gas is produced.
Showcasing the model
The goal of this model is to make a N.I.N.O that is playable. The first step is to characterize the micro-organism used. In this case the values used will come from the characterization of W3110 e. coli ‘ Wildtype’ strain13 .Table 1 shows the parameters that are used as input for the growth calculator. The italic parameters are easy adjustable, the non-talic parameters are inherent to the used micro-organism and its environment (temperature, type of medium, etc.).
Table 1: Key input parameters of the growth calculator and their values.
*Values come from table 3 and 4 of REFXXX
** Value is not in the report and is made up
When the values of are used as an input for the growth calculator the following graphs are returned (see figure 3). The top left panel shows the concentrations of glucose, biomass, acetate, and ethanol. At first the concentration of glucose is increasing because the rate of consumption is lower than the amount of glucose added by the feed. After approximately 6 hours this starts to change and consumption goes faster than the growth. This has to do with the increased biomass concentration, more cells means faster consumption of glucose. After 12 hours all glucose gets consumed before new glucose is fed into the reactor. This immediately has an effect on the growth rate (bottom left panel). At first the growth rate sky rockets to 80% of the maximum. When the glucose runs out the growth rate drops to zero until new glucose is fed into the reactor. The effect of this is also seen in the biomass concentration. It shows that the population is going into a stationary phase (plateauing). This is something that will increase the RpoS concentration in the cells which leads to the production of RFP.
Figure 3: Resulting graphs after running the growth calculator with the inputs shown in table 1.
Using this information you can deem the game unplayable. The next thing you do is turn some knobs of the design to see whether you can make the game playable. With a maximum growth rate of 0.44 it is probably difficult to make a game last 72 hours without getting into stationairy phase. So we limit ourselves to 1.5 days (36 hours). We will also increase the amount of sugar in the feed by increasing the feed volume per activation from 0.5 to 1ml, and decreasing the start volume to 10ml. This yields the following figure.
Figure 4: Results of the growth calculator after altering some design parameters.
Compared to figure 3, figure 4 has a longer period of exponential growth and its biomass graph is not plateauing. Combined with a larger feeding interval at the start and a smaller at the end you can argue that this game is playable. When a game is deemed playable you can now extract the design parameters from the calculator. In this example, the size of the offgas reservoir should be 3.5ml, the size of the feed reservoir should be 18 ml, the size of the reactor 28ml, etc.
Model assumptions and limitations
The model is not perfect. This is because there are some assumptions made and the model has its limitations as well. This paragraph explains the assumptions made and the known limitations of the bioreactor. However, it could be that there are some limitations that are unknown that decrease the accuracy of the model. In this case it is important to find out what is causing the drop in accuracy and try to solve it empirical.
The first assumption is that the pH is buffered well enough that the pH does not drop significantly because of the CO2 and acetic acid in the solution. A too low pH has big consequences for the growth and the stress levels of the micro-organism as well as the solvability of CO2.
The second assumption is that the maintenance of the micro-organism does not change. A rise in its maintenance (the energy needed to keep a cell running) can cause a drop in yield of biomass over substrate. In this model the yield is set at a certain value. The maintenance can rise because of certain inhibitors (like acetate).
Concept design
Figure 5 shows a concept design. In the first sketch, the concept design is depicted. It shows a symmetrical design. The reason for the symmetrical design is that it can be used in any way the user wants. The N.I.N.O. has two buttons: the start button to inoculate the reactor (green) and the feed button (blue). Both buttons will add liquid to the main bioreactor, increasing the volume inside (image 2). When the micro-organisms grow, they will start to provide feedback. If they are stressed, a red pigment (RFP) will be produced as shown in image 3. The fermentation of glucose (the substrate used) will result in the production of gas (CO2). This gas will inflate the flagella, thereby communicating to the player that they are alive and growing!
Figure 5: A concept of the physical design. The figure shows different phases of the N.I.N.O.
Project timeline;
Figure 6 shows the time-line of the project. The total project consists of multiple chapters. These chapters are.
- Transformation of the micro-organism
- Characterization of the micro-organism
- Biochemical process design (using the growth calculator)
- The design of the N.I.N.O
- Testing cycle
- Project delivery
Preparation before the start of the project
Preparations are necessary to make sure the project is off to a good start. These preparations include discussing the plan with experts to reduce the number of mistakes and do-overs. Reserving project requirements such as lab space. Ordering customized lab consumables such as DNA parts, primers, etc. Fixing all the hassle around the stay, f.e. a visa, a place to stay, etc.
Transformation of the micro organism
The first item on the to-do list is the selection [dv1] and transformation of the micro-organism. When selecting for an organism, the characteristics that are important should be considered. The strain should be easy to modify/transform, RpoS-expressing, and the maximum growth rate in an anaerobic environment should be in the right range. With the transformation, the red dye producing function is built into the genome of the organism. This red dye provides feedback to the player.
Characterization of the micro-organism
After the micro organism is chosen and the RFP gene is transformed into the organism, the strain needs to be characterized. Therefore, a batch fermentation is run which gives the necessary constants to be used in the growth calculator. These constants are µmax, KS, Kd, YX/S, YP,acetate/S, and YP,ethanol/S. The second purpose of the characterization is to see under what conditions and how much the RFP is expressed.
N.I.N.O. design and prototyping
After the micro-organism is characterized, the design and prototyping of the actual reactor can start. The physical design focusses on the usability and aesthetics of N.I.N.O. The final design should meet the requirements set out in the design section. The design of N.I.N.O. will be done parallel to the biochemical design (next paragraph).
Biochemical design
The biochemical design is made using the growth calculator. With the input from the characterization, the growth calculator is used to obtain the biochemical design. The biochemical design consists of the max volume of the reactor, volume of the feed reservoirs, feed volume per activation, start concentrations, and concentrations of chemicals in the feed.
Testing and improving
While the growth calculator and the characterization will mitigate the risk of failure in design, the system should be tested and tweaked to ensure that the game is playable. This is done in the testing and improving phase.
Project delivery
The testing and improving phase yielded a playable N.I.N.O. The deliverables of the project are made in the final week. The deliverables consist of a presentation, an N.I.N.O. prototype, and a blueprint of the N.I.N.O.
Figure 6: Project N.I.N.O. timeline.
Important milestones
The project consists of a couple of important milestones that, once reached, greatly increase the probability of achieving the ideal outcome. By listing these milestones in a chronological order, it is easier to keep track of the progress. These milestones are discussed in the following paragraphs.
Milestone 1: Successful transformation
The right micro-organism is selected, the necessary genes (as described here) are built into the organism, and expression is confirmed. This means that the micro-organism is ready to be characterized. Before this step is completed, it is impossible to start the next phase. When this milestone is achieved, the timetable (from figure 6) can be updated.
Milestone 2: Micro-organism is characterized
When the right micro-organism is available, the characterization of this organism can start. Characterizing the organism will yield the necessary parameters to model the growth of the organism. Subsequently, this model can be used to make the (biochemical) design of N.I.N.O. When this milestone is completed, the biggest hurdles in the project are overcome.
Milestone 3: The design of the N.I.N.O. working prototype is finished.
The third milestone is a finished design that meets all the requirements depicted in the design section. It is now time to make the working prototype.
Milestone 4 (final):Project delivery
The final milestone is the project delivery. All the deliverables are made and collected. Finishing this milestone means finishing the project.
project needs and costs;
The project needs resources to achieve the ideal outcome. Naturally, these resources come at a cost. Both the necessary resources and their costs are listed in this chapter. Note that the estimations of cost can diverge from reality due to the many unknowns, like lab work flow, salaries, and consumable costs.
Table 2: Estimated costs for project N.I.N.O.
A space in the molecular biology laboratory is required for the construction of the micro-organism and the assembly of the working prototype. It is estimated that the cost of this lab space is around $250 per week, resulting in a total of $3750 for the whole duration of the residency. For the characterization of the micro-organism, a batch and chemostat fermentation have to be run. This requires space in the fermentation lab. The cost estimates are $250 per week, with 4 weeks of characterization, the budget is set at $1000. While I’m familiar with all the necessary techniques to accomplish the goals, it is favourable to have some help in the lab to get started. Therefore, the project needs two lab technicians for the molecular biology lab and the fermentation lab. The cost of their time is estimated to be $8000-$10000 a month, meaning that for 40 hours $2000 should be sufficient. Because the methods of transformation and characterization are relatively cheap in terms of consumables, the costs are projected to be around $1500. To build the actual N.I.N.O. prototype, materials and third-party services are required. The budget is set at $3000. To bring this project to a successful end, some input from experts is needed. Input from an organism engineer is very helpful for the selection and construction of the micro-organism strain. Because the transformation is relatively simple, only 20 hours of help is budgeted at a cost of $2000. The same input is helpful in the characterization phase. Therefore, a fermentation scientist is budgeted at $2000. This leaves us with a total project budget of $17.250.
the future;
The objective of the N.I.N.O. is to connect people with biotechnology, connect people through biotechnology, and showcase biotechnology. All of these goals will not be met with just a working prototype. That is why Project N.I.N.O. has a section about its future.
From working prototype to version 1
To be able to achieve the stated goals the N.I.N.O. has to be produced in greater quantities. This means that, based on the working prototype, a first version of the N.I.N.O. has to be made. Some minor deviations have to be made to the design and a manufacturer needs to be chosen.
The use of N.I.N.O. version 1
There are many places where N.I.N.O. version 1 can be distributed. In this section, three options are discussed: in the classroom, at conferences, and for individuals.
Classrooms
In classrooms, the N.I.N.O. could serve as an interactive method of learning about microbiology. It can be used by teachers to learn the principles of microbial growth and GMOs to their students. The students can then apply their newly acquired knowledge to their N.I.N.O. to nurture their micro-organism.
Conferences
The N.I.N.O. could be the perfect gadget to hand out during a conference. It showcases what Ginkgo can do and will definitely attract the attention of clients and suppliers. It is possible to configure the N.I.N.O. in such a way that it can be played for a shorter period of time, such as a weekend or even just one day. A good example of a conference is the IGEM competition or SynBioBeta.
Individuals
For biotechnology enthusiasts, the N.I.N.O. can be available in a webshop. These individuals can then buy the N.I.N.O. at the cost price. This way, people can play N.I.N.O. with their family and friends or find similarly minded folks via the N.I.N.O. community.
The N.I.N.O. community
To connect people through the N.I.N.O., a community is established. A Reddit page is created where people can start threads, ask questions, or show what they are doing with their N.I.N.O. The reddit page now contains a couple of example questions and topics.
Future versions
The N.I.N.O. blue print is open source, and the design of the N.I.N.O. makes it modular. This makes it possible for people to add modifications themselves. People could install a bigger bioreactor in their N.I.N.O. or use their old N.I.N.O. to inoculate a new N.I.N.O. They could even change their configuration into a chemostat. Changing micro-organisms will be a bit more difficult since it needs a particular skillset to construct a strain that gives feedback to its user. It is, however, not that difficult for (PhD) students to develop an organism for N.I.N.O. themselves.
Disclaimer;
This project is a proposal and the details of this project are not etched in stone. This means that the details are still open for discussion. The provided details are merely the starting point for a discussion. Especially, the project scope, project timeline, and design requirements are up for debate.
references;
3. S. Johnson, in Wonderland: How play made the modern world, page 6 (Pan Books, London, 2018).
5. A. Wise, (PDF) sequences in the -35 region of escherichia coli rpos-dependent ... (1996)
7. E. Yohannes, D. M. Barnhart, J. L. Slonczewski, Ph-dependent catabolic protein expression during anaerobic growth of escherichia coli K-12. Journal of Bacteriology. 186, 192–199 (2004), doi:10.1128/jb.186.1.192-199.2004.
https://2019.igem.org/Team:TU_Eindhoven/Contribution