Difference between revisions of "Team:UCL/Entrepreneurship2"

Executive summary

1. Prepare restriction digestion mixture:
   IDT gBlocks
   - 10 ul of DNA (10 ng/ul)
   - 2 ul of 10 x 2.1 buffer
   - 0.3 ul of EcoR1
   - 0.3 ul of Pst1
   - 7.4 ul of milliQ H2O
   
   
   Other digestions
   - Required amount of DNA
   - 1 ul of 10 x 2.1 buffer
   - 0.3 ul of EcoR1
   - 0.3 ul of Pst1
   - add milliQ H2O up to 10 ul
   (if using total volume greater than 10ul, increase the amount of buffer accordingly)
   
   
2. Incubate at 37C for an hour
3. Heat inactivate by incubating at 80C for 20 minutes
4. Run a sample of digested DNA on a gel in order to confirm digestion:
   - 2 ul of DNA
   - 1 ul of 6 x Gel Loading Dye
   - 3 ul of milliQ H2O
5. If digestion is confirmed, proceed to ligation

Product

Overview

ProKao is a delicious chocolate bar that combines the goodness of chocolate and the benefits of probiotics. Our chocolate is sourced from organic and fair trade producers, and has been enhanced with billions of friendly bacteria, which have been shown to interact with your gut-brain axis, to make you happier and healthier, and help you cope with the stress of everyday life.

Once a day you will be able to indulge in our delicious chocolate bar, knowing that not only it will deliver these healthy bacteria to your gut, but also contains dietary fibers (prebiotics), which stimulate the proliferation of your natural microflora, without any added calories.

How our product came to be

The idea to create a probiotic to help with stress, anxiety and other mental health-related issues came from the core theme of our project: the gut-brain axis. Our original idea was to create genetically engineered probiotic strains but soon we realised that the current legislation doesn’t allow for the commercialisation of such products aimed at the general public, and only recently have tests with animals began taking place for pharmaceutical applications.

A three tier vision

The set of constraints we were initially faced with lead us to pivot away from focusing genetically engineered probiotics from the start. Instead we opted for the following timeline:

1. A product based on characterised strains and other GRAS (generally recognised as safe) ingredients that have a track record of efficacy and clinical data to back them up. This will be our minimum viable product, categorised as a food.
2. Once the mechanisms behind the effect of different microbiome profiles have been fully identified it will be possible to create enhanced strains. This can be done without necessarily being subjected to the tight regulations that restrict genetically engineered products, of course this would limit the scope of our action to certain techniques (deregulation, generating mutations, directed evolution, CRISPR-cas9). Another option would be to run our own experiments on particular strains we create or discover and patent them. These products would also be characterised as foods.
3. Using synthetic biology methods to enhance the production of particular compounds, introduce the genetic circuits that we have created to respond to environmental triggers and create ways of controlling the production of particular substances. This phase offers the greatest potential and the widest variety of applications; bacteria can be engineered into microbial factories that produce pharmacologically active substances of our choosing and secrete them in the desired amounts. A product such as this one is beyond the scope of what current legislation allows, and would only be considered for approval as a therapeutic and therefore be subjected to clinical trials.

Why Chocolate?

Independent studies have shown chocolate to be 3 to 4 times more effective in delivering probiotics to your gut than dairy products, as it protects them from being degraded in the passage through the stomach.

It is a delicious treat that will not only benefit your gut flora, but also has all these added benefits:

• Reduces the risk of cardiovascular disease

There are hundreds of studies that provide supporting evidence that chocolate improves the functioning of the cardiovascular system, due to its high content in antioxidants.

The flavanols in dark chocolate have been shown to increase the production of Nitric Oxide (NO), which makes the arteries relax, lowering blood pressure, and facilitating blood flow (Fraga, 2011). This also improves athletic performance, and recovery due to its anti-inflammatory properties.

In addition to this, chocolate promotes the creation of good cholesterol, which in the long term reduces the cholesterol accumulation in the arteries. (Buijsse, 2006)

• Improve Brain function and enhance mood

Dark chocolate contains compounds such as anandamide, PEA, theobromine and tryptophan, that increase serotonin and endorphin levels, which, in combination with our added probiotics, boost overall mood, and make you feel happier by encouraging the release of certain neurotransmitters such as melatonin. The consequences are increased libido, and an antidepressant effect.

The high flavonols content also improves memory and cognitive function, allowing you to be more aware, keep a sharp mind and a positive attitude (Scholey, 2013)

• It contains a high concentration of minerals

  100g of Quality Dark Chocolate (70-85%) contains:

  - 67% of the RDA for Iron.
  - 58% of the RDA for Magnesium.
  - 89% of the RDA for Copper.
  - 98% of the RDA for Manganese.
  - plenty of potassium, phosphorus, zinc and selenium.

  These are essential in building muscles, optimizing energy production, and are necessary for your immune function.

References

• Buijsse, Brian, Edith JM Feskens, Frans J Kok, and Daan Kromhout. 2006. Cocoa intake, blood pressure, and cardiovascular mortality: the Zutphen Elderly Study. Archives of internal medicine 166, no. 4: 411-417.
• Fraga, César G, María C Litterio, Paula D Prince, et al. 2011. Cocoa flavanols: effects on vascular nitric oxide and blood pressure. Journal of clinical biochemistry and nutrition 48, no. 1: 63
• Scholey, Andrew, and Lauren Owen. 2013. Effects of chocolate on cognitive function and mood: a systematic review.Nutrition reviews 71, no. 10: 665-681.

Intellectual property

1. (If using part from the distribution: resuspend the DNA in 10 ul of MiliQ water, making sure that it turns red. Wait 10 minutes before adding the DNA to cells)
2. Put a tube of NEB DH 5 alpha E. coli cells on ice and wait until they thaw completely. Divide the cells into 50 ul aliquotes.
3. Add 1 ul of plasmid DNA to 50 ul of cells.
4. Mix by carefully flicking the tube. Do not vortex or pipette in and out!
5. Place the mixture on ice for 30 minutes.
6. Heat shock the cells at 42 °C for 30 seconds and immediately put on back on ice.
7. Keep cells on ice for next 5 minutes. Do not mix.
8. Pipette 950 ul of SOC media kept at room temperature into the mixture. If SOC is not available, use LB.
   
9. Incubate the mixture at 37 °C for 60 minutes
10. Prepare plates with appropriate antibiotics. Bring plates to room temperature before plating. Use 2 plates per transformation reaction.
11. Plate 200 ul of cells on one plate.
12. Pellet the remaining cells and resuspend in 200ul of LB.
13. Plate the remaining cells on second plate.
14. Incubate plates overnight at 37 °C.

Manufacturing Operations

Prototype

With our product implementation plan in place, and a solid business model, we have developed a prototype with our engineered bacteria to test how effective the chocolate matrix is in encapsulating the bacteria and ensuring their survival for an effective delivery.

Protocol

1. Innoculate in two different Falcon tubes Nissle- TPH1 20ml of LB + Cam and shake overnight at 250rpm and 37ºC
2. Centrifuge at 40000rpm for 7min and discard the media.
3. Resuspend the pellet of one of the tubes in a solution composed of 1g of sugar and 1ml of miliQ water (A), and the one of the other in 500ul of miliQ water (B)
4. Melt 50g of dark chocolate (52%). The product also contained: Cocoa butter, Whey powder, Soya Lecithin, Polyglycerol polyricinoleate, and vanilla extract.
5. Mix 10g of chocolate with 500ul of the solution A (labeled 1.50 in the photo), and solution B (labeled 2.50).
6. Mix 10g of chocolate with 250ul of the solution A (labeled 1.25), and solution B (labeled 2.25)
7. Mix 10g of chocolate with 50ul of mili Q water for the Control

Note: the chocolate must be at under 40ºC before mixing with bacterial solution

8. Leave samples in the fridge overnight



9. Cut 5g of sample and wrap in aluminum foil for storage, to test shelf life



10. Cut 2g of sample and add to 5ml of LB in a falcon tube
11. Water bathe falcon tubes at 39ºC until the chocolate melts and mix by vortexing until a homogeneous texture is achieved
12. plate 100ul of solution in Cam LB agar plates and incubate overnight at 37ºC

The bacteria grew back on the agar plates after having been placed on the chocolate matrix overnight, which showed chocolate is an adequate media to place them. In addition, we observed a higher number of CFU in the plate of solution B, suggesting the more effective method for resuspension of the pellet.

However, from literature we know that the delivery and shelf life is best if the bacteria are microencapsulated beforehand with methods such as spray drying. There is also literature showing that when tested in a simulator of the human digestive system, probiotics in a chocolate matrix proved to reach the gut more effectively than those in Dairy (Possemiers, 2010)

This is the first step towards a more sophisticated prototype, in which we will test freeze dried bacteria against other types of microencapsulation, to optimize the delivery, as we want to be able to guarantee that at least 2 billion CFUs reach the gut and colonize it.

Our finalized chocolate bar will be a 50g single dose rectangular bar.

Scale-up Production

Manufacturing will be facilitated by outsourcing the production of our ingredients to key partners with experience in the sector. These partners will provide the already processed chocolate, and encapsulated probiotics. They will be selected according to the quality criteria with which the product identifies. This is mainly organic fair trade cocoa pods, and other more specific certifications to cater to the needs of specific sectors of the general public, such as a gluten free certification or dairy free.

This will be beneficial to us, as these companies can off-set early production, which will allow our products to reach the market sooner. This allows us to focus on the further research of the biological components needed for the development of the company to the second and third stages.

For the first stages of the business development, we would use strains whose effect is documented in literature

Production will take place following two different models, for two slightly different products

Stratified

In the stratified model, consumers identify themselves with a specific category, and select the chocolate bar that is more adequate to suit their needs.

Whereas most probiotics companies we found offered a range of different products based on flavours we would target market segments instead. We will produce a fixed number of bars with specific probiotic contents, specifically designed for each market segment. These will be differentiated in terms of age, sex, and lifestyle; which will lead to strata like: higher protein content for more athletic people, or a limited amount of lactobacillus strains for vegans.

This model has a simpler production, given that it’s a repetitive process in which machines can be programmed for a certain amount of hours to produce one specific type of bar, and then switch to a different one by just changing the proportions to be added, and producing a great amount.

Personalised

The personalized model, adds a layer of complexity to the structure. It will consist of made to order individual batches of probiotic chocolate tailored to the users goals and needs to in a way that has never been done before.

Using information supplied to us by the user we could design a probiotic fit specifically for their profile and monitor its effect by monitoring the state of the microbiome periodically. Over time more metrics and applications would be added, optimizing our ability to cater for each specific profile, and ultimately fulfilling the promise of power over all the potential that microscopic factories living within us can offer.

References

• Bravo JA, Forsythe P, Chew MV et al. (2011) Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proc Natl Acad Sci USA 108, 16050–16055.
• Dapoigny M, Piche T, Ducrotte, Lunaud B, Cardot JM, Bernalier-Donadille A (2012): Efficacy and safety profile of LCR35 complete freeze –dried culture in irritable bowel syndrome: A randomized , double blind study. World J Gastroenterol 18:2067-2075.
• Desbonnet L, Garrett L, Clarke G et al. (2008) The probiotic Bifidobacteria infantis: an assessment of potential antidepressant properties in the rat. J Psychiatr Res 43, 164–174.
• Hsiao EY, McBride SW, Hsien S et al. (2013) Microbiota behavioral and physiological abnormalities associated with neurodevelopmental disorders. Cell 155, 1451–1463.
• Ko CY, Lin HTV & Tsai GJ (2013) Gamma-aminobutyric acid production in black soybean milk by Lactobacillus brevis FPA 3709 and the antidepressant effect of the fermented product on a forced swimming rat model. Process Biochem 48, 559–568.
• Messaoudi M, Lalonde R, Violle N, Javelot H, Desor D, Nejidi A, et al. (2011): Assessment of psychotropic-like properties of a probiotic formulation (Lactobacillus helveticus R0052 and Bifidobacterium longum R0175) in rats and human subjects Br J Nutr 105:755-764
• Possemiers, Sam et al. (2010). Bacteria and chocolate: a successful combination for probiotic delivery."International journal of food microbiology 141.1: 97-103.
• Rao AV, Bested AC, Beaulne TM, Katzman MA, Iorio C, Berardi JM, Logan AC (2009): A randomized, double-blind, placebo-controlled pilot study of a probiotic in emotional symptoms of chronic fatigue syndrome. Gut Pathog 1:6.

Business Environment

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1. Digest at least 500 ng of each part according to the restriction digestion
2. Run the digested DNA on the gel according to gel electrophoresis protocol
3. Identify the parts that you want to ligate on a gel and cut the bands out using razor blade
4. Purify the excised bands using the commercial kit according to the manufacturer's instructions
5. Quantify the DNA yield using DNA nanodrop
6. Proceed to ligation

Marketing

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1. Prepare the PCR mix:
   - 12.5 ul of 2 x Q5 PCR master mix
   - 1.25 ul of 10 uM forward primer
   - 1.25 ul of 10 uM reverse primer
   - 2 ng of DNA to be PCRed
   - add milliQ H2O up to 25
2. Set up the PCR cycles according to the following rules:
   Initial denaturation
   - 98C for 30 seconds
   35 cycles
   - 98C for 10 seconds
   - 30 seconds at primer melting temperature
   - 72C for 30sec/kb of PCRed fragment
   Final extension
   - 72C for 2 minutes
   - Hold at 4C
3. Confirm the PCR by running 2 ul of the product on the gel according to the gel electrophoresis protocol

Management

1. When designing the gBlock fragments for Gibson Assembly, make sure that the fragments have ~20 bp overlap and that first and last insert fragment have ~20 bp overlap with respective ends of PSB1C3
2. Convert the concentration of vector and inserts from ng/ul to pmol/ul using the following formula:
   
3. Prepare the Gibson Assembly mixture:
   - 0.08 pmol of each insert
   - 0.04 pmol of vector
   - 10 ul of Gibson Assembly mix
   - add milliQ H2O up to 20 ul
4. Incubate the reaction at 50C for 15 minutes. Following incubation, put samples on ice.
5. Proceed to transformation protocol. Use 2 ul of the Gibson Assembly reaction mixture for transformation

Financial Data

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