Bioproduction

Part1 : Tele-Experiment Homework

Design and run an experiment to understand how different factors influence bioproduction.

The available factors are genes (pAC-LYC, pAC-BETA), media (LB, 2YT), concentration of precursor (fructose), temperature, and shaking. Please follow the steps below to execute the homework:

  • A) Design the experiment

  • B) Create the code for the tele-experiment on the robot

  • C) Run the simulation to make sure that there is no syntax error

  • D) Schedule a time with Pat for running the code on the robot!

Part 2: Identify a cool gene (related to your final project)

Identify a cool gene (related to your final project) and try to order it via twist bioscience following the video instruction bellow. Please select "Clonal Genes" for the gene type. Once you finished uploading your gene sequence, select a vector (please refer toSebastian's slidefor more information), and perform codon optimization (if necessarily), please download the GenBank file of your gene (+ vector) and send it to here :https://drive.google.com/drive/folders/1yX2VgEaWOb29Kx-B_jswn39Zx-m8N3Vx?usp=sharing

Part 3: If you have the capability of Gingko Bioworks foundry...

Write a short paragraph responding to "If you have the capability of Gingko Bioworks foundry, what would you do and why?" These could be COVID19 related projects or broader synthetic biology projects. Ginkgo Bioworks is taking proposals to leverage the use of their platform to support technical projects; how might you leverage Ginkgo’s technical platform to support your project? Write a description of how you would utilize their platform. What tools and capacities would you use? How would you use them? How would the use of Ginkgo’s platform accelerate or increase the technical capabilities of your project?

MIcrofluidics

Part 1: Design a milli-fluidic device for microbial cell-culture

In this homework, we will use Autodesk Fusion 360, a 3D CAD software, to design a 3D printed micro/milli fluidic device. Please install the software using the following instruction.


Part 2: Design experimental conditions for...a microbial cross-feeding interaction?

  • Find a research or journal article where researchers cultivate 2 or more microbial strains. What technology are they utilizing? How scalable is this approach to more than 2 strains? How do they address issues related to requiring multiple media?

  • Propose a technology for culturing 2 or more strains. How might you innovate in this area given the paper you reviewed?

  • One of the great challenges in microbiology currently is culturing “unculturable” microbes. Propose a methodology for how you might explore this significant space of uncultured microbes.

  • Review an article on an artificial gut-on-a-chip technology. What scientific hypotheses are they testing with this in vitro tool? Could you propose an upgrade or innovation to their technique to enable the exploration of other scientific hypotheses? Provide an example of at least one hypothesis you would explore with your proposed system.

  • One of the biggest challenges in public health is quickly detecting new disease outbreaks. How would you go about adaptively responding to new outbreaks? Biobots is using qPCR, so you need to know what you are looking for. How might you develop a technology with a more general view? Some example approaches include microfluidics and point-of-care sequencing, but what else? In particular, are there ways to look for RNA viruses like the flu and SARS-CoV2?

  • Extra credit: In the hardware class you designed a fluidic device. Please either use this device as a starting point, or provide a sketch design of a fluidic device that could be used in the gut-on-a-chip system you are proposing in question 4.

Final Project ideas

Initial ideas

Idea 1: Bioprinted Multilayer Dynamic Material

  • Layer 1(bottom):Iridescent layer

    DNA origami to create static nanostructure that can assemble into dynamic microstructures that create the iridescent layer

  • Layer 2(middle): Pigment Layer

    Different pigments could be expressed in response to different conditions/stimuli

  • Layer 3(Top): Melanin Layer

    Reversible proteins that can toggle between 2 states controlling how much melanin is expressed.This enables different degrees of light to pass through, therefore exposing a range of pigments and iridescence

 
CEPH_diagram.jpeg

Idea 2: Freeze dried personalized nutrition and flavorings for astronauts (Deep Space Food Challenge)

  • Freeze dried cell free systems to produce micro, macro and phytonutrients + flavors and smells on demand

  • Advantages:

    • High shelf life

    • Compact/easy to store

    • Customizable to each astronaut’s needs and preferences

    • Safety: Doesn’t require live organisms

    • Enhances the food experience + offers more variety

3D bioprinting and biofabrication

Pre-homework questions:

  • From an ethical and moral standpoint, list pros and cons of working with hiPSCs as opposed to embryonic stem cells?

  • If we are to use organoid models as surrogates for in vivo tissues, what type of functional attributes would be critical to add/engineer into such organoid systems?

  • Can you think of any limitations in current organoid and or organ on a chip systems in recapitulating human biology?

  • If you were to theorize an experiment about improving current organoid models, which aspects of the microenvironment would you consider adding beyond chemical?

  • In the tissue engineering paradigm, what properties are important to provide from a scaffold and bioreactor?

The homework component will include:

  1. Read prescribed articles and answer questions.

  1. Live streamed treatment of EHTs with student input of which drugs to use, reasoning behind selection of drugs (what do each of them do?), concentrations to use and if incremental dosing is required?

Aromata

on demand bio-production of aromas to enhance flavors and food experience in long-duration space exploration

 
 

Project abstract

Aromata is a personal wearable olfactory interface that leverages synthetic biology to enable astronauts to safely and conveniently produce aroma compounds on demand. These aromas are customizable based on preference, and could enhance the astronaut’s experience of flavor in space. Moreover, different aroma combinations could be paired with the crew’s limited menu, altering the ‘perceived’ flavor of the same foods; therefore, providing astronauts with more variety and novelty during long-duration missions. 

Using freeze-dried cell-free (FD-CF) systems that take little space and are shelf-stable at room temperature, Aromata offers a novel approach to augment the quality and expand the variety of flavors, using minimal inputs and resources, and creating minimal waste. 

Background

Food in space

With long-duration human exploration missions to Mars and beyond on the horizon, there’s an increasing need for novel food production technologies or systems that require minimal resources and produce minimal waste, while providing safe, nutritious, and tasty food for the astronauts.

Eating is an important part of crew morale and the one communal time when they share a meal together. However, astronauts found that their taste buds did not seem to be as effective when they were in space. This diminished perception of tastes and smells make food less appetizing and contribute to insufficient food intake and calorie deficit in astronauts.

Since chemosensors play a key role in the control of food intake, changes to the odor and/or taste of a food may affect its intake.

giphy (19).gif



Aroma and flavor

Flavor is a complex perceptual impression of food or other substances. It consists of sensory information about taste, smell, texture and temperature. However, Of the three chemical senses, smell is the main determinant of a food item's flavor.

giphy (15).gif

Five basic and universally recognized tastes include sweet, sour, bitter, salty and umami (savory) . The number of food smells, on the other hand, is unbounded; a food's flavor, therefore, can be easily altered by changing its smell while keeping its taste similar.

 
slide1-beef-how-to-pair-flavors.jpeg
 
 

Flavorings are edible chemicals and extracts that alter the flavor of food and food products through the sense of smell. Three principal types of flavorings used in foods include natural flavoring substances, nature-identical flavoring substances and nature-identical flavoring substances.

Due to the high cost, or unavailability of natural flavor extracts, most commercial flavorings are "nature-identical”, which means that they are the chemical equivalent of natural flavors, but chemically synthesized rather than being extracted from source materials.

Alternatively, metabolic engineering (including enzyme technology) offers a very promising option for natural flavor biosynthesis. Both genes and enzymes involved in the biosynthesis of flavors are constantly identified, which provides insight into metabolic engineering of flavor compounds (i.e. aroma). The use of enzyme-catalyzed reactions provides higher stereoselectivity than chemical routes. It is also more cost effective and has far less negative environmental impact.

Innovation

Aromata incorporates the use of freeze-dried cell-free systems, and a built-in mircofluidic bioreactor to produce controlled amounts of aroma compounds on demand.

Freeze-dried cell-free systems

Although superior to chemical synthesis, metabolic engineering of microorganisms for flavor(aroma) compound production could be time-consuming, and involve a non-intuitive, combinatorial tuning of biosynthetic pathway variations to meet design criteria. Cell-free systems (CFS) could resolve many of these complexities, allowing for faster design–build–test cycles and direct manipulation of the reaction.

18-9-Melinek-opener.jpeg

Components of a cell-free protein

synthesis reaction: (extract, supplements, and a DNA template) with the key reactions that occur when they are combined.

Melinek et al., “Toward a Roadmap for Cell-Free Synthesis in Bioprocessing.”

 

In addition, by compressing buffers, cellular machinery, and molecular instructions all into a single freeze-dried (FD) reaction pellet, astronauts would be able to activate aroma compounds in space on-demand, only by adding water within a few hours, and without the need for specialized equipment and skill. 

 

BioBitsTM kits: Freeze-dried educational kits.

A) FD-CF demonstrations require only the addition of water to the supplied reactions and incubation for 1 to 20 hours at 25° to 37°C. (B) With the DNA template and any substrate molecules provided with the FD-CF reaction, the students just have to add water to run a number of bioscience activities.

Huang et al., “BioBitsTM Explorer.”

 

A cell-free approach was used to produce certain aroma compounds in the lab, proving that it is possible to produce a variety of compounds and develops protocols to produce more complex aroma combinations (aroma recipes).

 

Fragrance-generating enzymes as olfactory outputs

(A) Using FD-CF reactions, we manufactured enzymes that can generate various smells from the Saccharomyces cerevisiae acetyltransferase ATF1. (B) Production of fragrance molecules after substrate addition to overnight FD-CF reactions of ATF1, as detected by headspace GC-MS. Values represent averages, and error bars represent SDs of n = 3 biological replicates.

Huang et al., “BioBitsTM Explorer.”

 

Microfluidic Bioreactor

A microfluidic bioreactor inside the device incubates the CF systems, once they’ve been activated with water.

HCI

Specific aims

  1. Express a single aroma compound

    1. in E. coli

    2. in a cell-free system

  2. Create an aroma pack: a complex food aroma recipe

    1. Multiplexing multiple pathways in one cell-free system

    2. Combinatorial approach

  3. Design and prototype a user centric device, as well as method of use and operation tailored to the needs of astronauts