iGEM 2016 — Team Leicester

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# Team:Leicester/Notebook

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* ☰

Team Leicester iGEM 2016

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#### (Calcitonin Hyper Expression using E.Coli in Space Exploration)

* [What is Osteoporosis?](#whatisosteoporosis)
* [How does loss of bone density occur?](#lossofbonedensity)
* [How does this occur in astronauts](#occurinastronaunts)
* [Current Treatments](#currenttreatments)
* [Our Project](#ourproject)

Long term space flight is quickly becoming technologically possible, however the limiting factor will soon be the human body’s ability to survive the flight.l. Space exploration takes a toll on the human body and comes with many risks. A major health issue that arises with space exploration is Osteoporosis.

# What is Osteoporosis?

Osteoporosis, more commonly known as brittle bone disease, is a condition that causes the bone to become more porous due to a decrease in bone density. This results in the bone becoming more fragile, which can increase the risk of bone fracturing [1]. Studies have shown that the human body not being under the influence of gravity causes the bones to lose density at a rate of over 1% per month. The elderly on Earth lose bone density at a rate of 1-1.5% per year, so a new and more efficient treatment is sorely needed in both circumstances [2].

# How does loss of bone density occur?

Calcitonin is a hormone that strongly inhibits Osteoclast function. Osteoclasts are involved in the breakdown of bone into calcium, whereas Osteoblasts increase bone density [3]. The parathyroid hormone inhibits Osteoblast function and stimulates the activation of Osteoclasts. Calcitonin opposes the effects of this by lowering calcium levels via suppression of Osteoclast function and stimulating Osteoblasts [3].
After the age of 35 years, the ageing process occurs which results in difference in levels of Osteoclasts and Osteoblasts. This results in loss of bone density. This change in the bones results in the bones becoming more fragile in old age and the risk of fractures becoming more common, additional effects occur, such as the increased risk of kidney stones, due to the elevated levels of calcium in the bloodstream.

# How does this occur in astronauts?

The answer is simple: lowered gravity. A typical space mission forces astronauts to endure three fields of gravity, all with different strengths: the travel to the destination is where astronauts are weightless. On the surface of their destination, the gravity acting upon the body is far less than as if you were on Earth, and finally when you return to Earth, the body has to readapt to the gravity here [4]. The body is constantly adjusting to suit its environment, this means that when in an environment where there is a reduced gravitational field, it adjusts, in this case the stress that bones normally go through every day supporting our bodies is reduced/ removed. This means the body does not need the bones to be as strong, and so reduces the bone density. This becomes a problem when transitioning to an environment that has a stronger gravitational field than your body has adapted to, say when astronauts return to Earth after a long time in space. The changes that the body has to go through in order to adjust itself to each situation causes bones to lose density at over 1% per month[4]. Even though when astronauts return to Earth they go through rehabilitation, there is a risk that bone loss may not be fully corrected, therefore astronauts are at greater risk of Osteoporosis-related fractures in the future.

# Current Treatments

The most common treatment for Osteoporosis is calcitonin, however it is not very efficient for space travel due to weight.

Calcitonin is usually administered in 2 ways: orally or via a nasal spray. The calcitonin used in both methods are from salmon. Although it can be effective, there is a 50% difference in the protein sequence between human and salmon calcitonin, this could mean that the salmon analogue is not as effective, also, salmon calcitonin is known to cause nausea and vomiting [5]. Furthermore, calcitonin nasal sprays are ineffective and slow acting while oral administration does not work effectively as the peptide needs to be absorbed into the bloodstream in order to work properly [5].

# Our Project

![Cas9 Componwnts](https://static.igem.org/mediawiki/2016/thumb/5/58/T--Leicester--figure1cas9.png/747px-T--Leicester--figure1cas9.png)

We planned to use the CRISPR associated protein 9 (Cas9) protein, however it is a mutant, and is known as dCas9 targeted to the human Calcitonin promoter region in order to increase and decrease the expression of downstream elements. The dCas9 has a mutation in amino acid 840 causing the substitution of an alanine, this mutation cause the Cas9 to be unable to cleave DNA, instead the dCas9 will bind to the DNA and remain there until it is removed. To target the dCas9 to the promoter region we are using synthetic gRNAs, these are short scaffolds of RNA that have the complementary sequence to your target site. We designed 2 gRNAs targeted to a similar region of the calcitonin promoter,with complementary sequences 20 base pairs long, as this was the recommended length.

It would be technically very complex to assay the production of calcitonin in the E.coli, so therefore we decided to insert the calcitonin promoter into a GFP reporter setup, this comprised of the same elements in each track, but in different orders

![](https://static.igem.org/mediawiki/2016/d/df/T--Leicester--LeicesterLogo1.png)