1. Lactose Intolerance
By: Chloe Darancou

2. What is lactose intolerance?
Lactose intolerance is a condition in which a person cannot properly digest any products containing lactose; dairy products contain the lactose sugar that troubles people. The victims effected produce no lactase enzyme that breaks down the lactose into glucose and galactose; glucose and galactose are sugars that the body can breakdown and can use as a source of energy. The Lactase enzyme is typically produced by the small cell within the lining of the small intestine.

3. Types of Lactose Intolerance
Primary Lactose Intolerance
Secondary Lactose Intolerance
Congenital or Developmental Lactose Intolerance

4. The Reason
Millions of people all over the world struggle each day because of lactose intolerance issues. The victims affected constantly take supplements to combat the effects, or they avoid dairy all together. The pills that are available require its patients to take the supplement previous to consuming dairy. The effects of consuming lactose while being lactose intolerant are: nausea, vomiting, diarrhea, gas, and bloating. Individuals possessing this ailment feel restricted and limit themselves when referring to their dietary plan.

6. The Design
A bacterial strain of E.coli will be inserted with a gene that produces the lactase enzyme. The E.coli will uptake a plasmid that contains a gene that codes for a protein that is an enzyme, this enzyme will digest the lactose sugar that is being consumed.
The bacteria must be able to with stand high pH levels of the stomach
The bacteria must be able to stay within the stomach, so it can multiply and divide.

7. Competing technologies
There are lactase enzyme pills that exist by containing a lactase enzyme that helps digest lactose sugars that are implemented within the gut.
There are also dairy products that contains a lactase enzyme, so the enzyme digests the lactose, as a result the effect of the lactose-intolerance isn’t felt.
The Lactaid supplements are effective, but are not very practical in ways of constant consumption

8. Engineering
The LCT gene contains the instructions for creating lactase
Extract a healthy cell from the lining of the small intestine, locate the LCT gene in the DNA within the eukaryotic cell, cut the fragment with restriction enzymes, then cut a plasmid in a basic strand of E.coli with the same restriction enzyme
Insert this recombinant plasmid back into the E.coli through Heat shock methods.
Allow bacteria to grow on an agar plate with high pH
Find a capsule, insert nutrients and colonies of bacteria inside the capsule, then allow patient to take this pill once every five years

9. Engineering
The bacterial cells that are engineered and placed into the gut will start multiplying and producing the lactase enzyme; the engineered cells within the gut assist the previously existing intestinal cells to digest the sugar lactose.

10. CRISPR Clustered regularly interspaced short palindromic repeats .
The CRISPR is programed to target a certain sequence, so it can replace that sequence with its own.
The Cas9 enzyme locates the target sequence and cuts the DNA then the cell’s repair mechanisms take over, so the non-homologous ends join; this joining often results in a mutations, the result is an inactive gene
The gene is inserted into the CRISPR plasmid, by lining up perfectly with the DNA strand; the end result is an incorporated gene within the plasmid.

11. CRISPR

12. How it works?
LTC gene is extracted through a heathy small intestinal cells→ The LTC gene is inserted through CRISPR techniques( a gene s first silenced, then a gene is inserted) → The LTC gene is expressed→ The LTC gene creates the lactase enzyme → the lactase enzyme breaks down the lactose sugar → the lactose is broken down into the sugars galactose and glucose, which are usable sugars for the body

14. Materials needed Strain of harmless E.coli Micropipette
Healthy intestinal cells
CaCl2 solution( if needed)
Heat bath ( if needed)
Ice ( if needed)
Bacterial nutrients
Agar containing a high pH
Cas9 protein
Buffer
Micro centrifuge

15. Expected results
Presence of E.coli
Presence of lactase 1

16. Advantages Capsule can be taken once, and it lasts for a span of years
Cheaper than constant purchase of pills
Easy to take and effective
Cheaper than having to buy products with lactase already in them

17. Funding
The Lact. Bact. is worth funding due to easy accessibility to all materials needed
Great for families with low-income
Cost effective and efficient

18. Potential problems
The bacteria could denature if the pH levels are too high
The bacteria could just pass through one’s system
It is possible that not enough lactase is produced to completely digest the lactose
If you throw up then the bacteria would exit along with the stomach acid it is living in.

19. Safety
All lab materials will be disinfected with an auto clave previous to use
All workers will take extreme caution in measurements
All workers are required to wear a lab coat, goggles, and gloves.

20. Environmental risks
There are no serious environmental risks posed with this engineered bacteria
If the bacteria were to be released into the wild, then there would be no harm
The only possible harm that could arise is when CRISPR techniques are utilized

21. Are the risks worth the rewards?
The Lact. Bact. Will improve the dietary limits for those who are challenged with this ailment
There are no direct environmental risks from this engineered bacteria
There might be public and media backlash due to genetic engineering being involved, since a majority of the public is uninformed, the product will be questioned of its ethicality.

22. Testing Lactose intolerance test Hydrogen breath test
Stool acidity test

23. Testing
Testing can help determine the amount of bacteria needed within a tablet
Testing can determine how often the pill is needed to be consumed
Testing can help determine the effectiveness of the pill
Testing can reveal the sustained threshold of the bacteria within the high pH levels of the stomach
If the testing reveals that this bacteria optimally functions the way intended, then were can solve other intolerant issues.

24. Sources https://ghr.nlm.nih.gov/gene/LCT
tests
diagnosis/CON

25. Thank you!