BME494 Project Group7

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Contents

ABSTRACT

The GULO gene in mice is still active. Unlike humans, mice can produce their own vitamin C.
The GULO gene in mice is still active. Unlike humans, mice can produce their own vitamin C.


Vitamin C deficiency, while uncommon, causes scurvy, lack of immune system development, delayed natural healing ability, and weakness of bones and muscles. The human body was once capable of producing its own vitamin C via the GULO gene, now dormant in the human genome but active in that of mice. The active GULO gene produces L-gulonolactone oxidase, a catalyzing enzyme necessary for the production of ascorbic acid found in the liver or kidneys. This project aims to control GULO gene activation in mice using an infrared light switchable protein, and to encourage similar studies in the human genome to benefit those most at risk for vitamin C deficiency.






BACKGROUND

Symptoms of scurvy, caused by vitamin C deficiency. Although very treatable, the disease still occurs in poverty-stricken countries.
Symptoms of scurvy, caused by vitamin C deficiency. Although very treatable, the disease still occurs in poverty-stricken countries.

This project has the potential to reduce vitamin C deficiency in humans living in environments where ascorbic acid-rich foods are sparse. Health benefits extend beyond third world countries – studies have shown a possible link between ascorbic acid and the selective killing of cancer cells. Self-production of vitamin C may therefore provide a valuable immunodefense weapon against cancer development in addition to benefiting overall health. Additionally, reactivating a dormant gene has implications for other disused parts of the genome. If successful, this project might encourage the re-activation of other dormant useful genes (in human genomes or otherwise). Finally, the attachment of a light-switchable protein to GULO can be altered so that it is applicable to other genes, providing a working toggle switch for any compatible function.







PROOF OF CONCEPT DESIGN

  • New Natural Part: PIF3-YFP
The GULO gene is attached to the YFP protein. Light will cause the protein to attach to the PhyB protein, activating the GULO gene.
The GULO gene is attached to the YFP protein. Light will cause the protein to attach to the PhyB protein, activating the GULO gene.

Explained in the research paper 'Spatiotemporal control of cell signalling using a light-switchable protein interaction' published by 'Nature' in October 2009, Photochrome Interaction Factor 3 (PIF3-YFP) is a protein that when activated by red light, attaches to Phytochrome (PIF-PhyB) and activates whichever sequence is attached to it, in this case, GULO. The attachment, and therefore the activation, is reversed when infrared light is used.

  • Key Pre-existing Part: GULO

The sequence for the GULO gene, responsible for the GULO enzyme that catalyzes vitamin C, was available on the Registry of Standard Biological Parts (www.partsregistry.org). The essential codons were extracted and the edited sequence was converted into a BioBrick using the following primers:
Forward primer: 5’ – GCCTTTCTTGGTACCTGTG – 3’
Reverse primer: 5’ – ATCTTTCAGAGTCTTTTAA – 3’

Assembly Scheme

The second plasmid contains the GULO gene attached to the YFP protein sequence. When red light is flashed on the petri dish, the PIF3 attaches to the PhyB sequence on the second plasmid, activating the GULO gene and beginning production of ascorbic acid.

The GULO gene, once attached to PIF3-YFP and activated with red light, will continue to produce vitamin C until the death of the E. coli bacterium. After cell proliferation, the gene is still present in the system but remains dormant until re-activation with red light. The activation is reversible with infrared light, as shown below.


TESTING


Measurement

Vitamin C Test Strips
Vitamin C Test Strips

The amount of vitamin C produced by the plasmids will be quantified using test strips. The color of the strips indicates the level of ascorbic acid. Pink indicates full strength, dark pink indicates half strength, light purple indicates 1/4 strength, purple indicates 1/8 strength, and deep purple indicates 1/16 strength. Pink is around 16 times the strength of the deep purple indicator shown to the right.


Expected Observations

The above graph shows the rate of production of vitamin C corresponding to the wavelength of light used on the bacteria. (Each color represents a different test, and roughly the same amount of E. coli was used each trial.) The YFP protein is activated at 650 nm, which is when vitamin C production begins. The protein is turned off at 750nm. At this point most of the trials have reached their peak and begin their descent. No increase in production occurs after 750 nm.
The graph below shows the concentration of vitamin C in the body over time. Vitamin C production begins when red light is shone. The first peak occurs when all the cells present at the initial flash die, ceasing production. As the body uses up the vitamin C, the concentration decreases until the next flash of light activates the offspring cells containing the dormant gene. Since the second generation of cells is larger than the first due to prime growth conditions, the peak amount of vitamin C concentration is greater than the first. The process repeats until it is determined that the body has received enough vitamin C supplement.


Tuning Our System
One variable to be tuned is the amount of protein interaction factors present in each cell. More factors leads to a faster rate of vitamin C production and a greater quantity of cells possessing the dormant gene within a time frame. The life of the cell, and the duration of time for which vitamin C is produced, remains unchanged.
Another is the lifespan/rate of reproduction of the E. coli, which is controlled by the bacteria's environment and the nutrients available to them. If the cells are allowed to grow freely, vitamin C concentration will follow the graph shown above in Expected Observations, and will increase each cycle. Otherwise, the number of cells can be kept constant if the rate of reproduction is equivalent to cell death rate – the concentration of vitamin C produced each cycle is kept constant.




In the graphs above, vitamin C production continues until all first generation cells die. The second peak occurs when all second generation cells die, etc. Vitamin C concentration decreases as the body uses up the vitamin C.

HUMAN PRACTICES

Overdose of vitamin C may be possible if the cells continue to grow in the body and are activated. In this case, vitamin C production can be stopped using infrared light. In the aforementioned experiments, flashes of red light were intentional at specific time intervals. Accidental activation of the GULO gene may occur if the patient is exposed to strong or invasive red light – targeted cells near the skin may activate while the patient is unaware of ascorbic acid production. Since vitamin C overdose is rarely fatal, the greatest danger is that the cells containing the gene grow outside of the targeted organ in an area it cannot be digested.

OUR TEAM

Larry Ngo
Larry Ngo
Hello! I am a 1st year Masters Student in Biomedical Engineering. I'm taking Synthetic Biology to hopefully get some ideas to apply to my Applied Project. I like pigs :]







Kris Phataraphruk
Kris Phataraphruk
I am studying Biomedical Engineering and this is my junior year at Arizona State University. I am taking BME 494 in order to expand my range of the different engineering fields in biology. I am hoping to get into the Master's Program at ASU and go into genetic or prosthetic engineering research.






Catherine Terrell
Catherine Terrell
Hello! My major is Biomedical Engineering. I am taking Synthetic Biology because I would like to know more about genetic/DNA engineering and whether it is a field I'd like to pursue. An interesting fact about myself is that I edit and write for a small honors magazine.




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