OXIDATIVE STRESS

After choosing our topic of healthy ageing we spoke to various researchers in the area, including Dr. Max Yun, a Senior Research Associate in the UCL’s Division of Biosciences who recommended a paper to us - ‘The Hallmarks of Ageing’. This highlighted not only is ageing a complex, multi-faceted problem, but also that it wouldn’t be possible to target all hallmarks, instead, we chose to focus on reactive oxygen species (ROS) which underpin some of the hallmarks (1) of ageing.

For instance, endogenous damage caused by ROS leads to DNA lesions including telomerase shortening(1). These lesions essentially lead to genomic instability (1). Ageing in this instance is caused by imbalance between DNA damage and DNA repair, for instance insufficient repair mechanism or excessive DNA damage promotes ageing (1).

Oxidative stress can also cause proteins to unfold and aggregate, again leading to ageing (1). We found greater strength in this argument when we were shown data from research undertaken at Imperial University showing oxidative stress to have a detrimental impact upon protein stability. The consequence of this is in turn reflected in protein activity and hence ageing. A description of these results is included below.

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Clearly the impact of oxidative stress upon protein structure is significant and since structure and function are inexplicably linked the functionality of the proteins are likely to be hindered too. Thus, we are looking towards preventative methods that will lessen the amount of oxidative stress that proteins are exposed to.

LYCOPENE CONCEPT

Lycopene is a carotenoid, and is the compound responsible responsible for giving the distinctive red colour to fruit such as tomatoes and watermelon. It is for this reason that lycopene has previously been used and characterised within iGEM as it lends itself to colorimetric detection. However, we believe this does not completely harness the power of this compound.

 Lycopene is one of natures most powerful antioxidants. This is due to the numerous saturated bonds within its structure which enables it to quench ROS including singlet oxygen (2). This provides our cells with protection against damage to critical biomolecules -which can ultimately lead to ageing. We are looking towards using this as a probiotic treatment as this enables us to make the use of UCL iGEM 2015s 'gut on a chip' to test the workings of this.

SYNTHETIC VS NATURAL

So why the need for synthetic biology? Why not just eat tomatoes or watermelon? Well, the synthetic form of lycopene has been shown to be more effective in neutralising this oxidative stress because it is in a more bioavailable form compared to the form that is found in these fruits (2). This enables the compound to be more readily absorbed and consequently more effective in minimising ROS damage.

DESIGN

>We combined the lycopene biobrick already existing in the registry with the NarK promoter in order to control the expression of lycopene. The biobrick falls into two parts:

1. Sensing The promoter senses oxidative stress and in response turns on transcription of the downstream genes.

2. Responding The gene for the antioxidant, lycopene is turned on as a response to oxidative stress. Lycopene-NarK initial design:

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Further design steps:

1. Removal of illegal restriction sites within the gene through silent mutations.

2. In order to minimise the time to receive DNA from IDT, design steps were taken in order to put these into gBlock format. This required splitting up of the rather large gene construct into 4 parts of around 500bp as well as consideration for the collating of the parts into one by Infusion.

The final outcome was a composite biobrick, BBa_K1954001, which we submitted to the registry.

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Experimental design:

We wanted to find validated protocols for the expression and detection of lycopene. Firstly we wanted to induce oxidative stress. After several options were considered, from nitrogen gas to hydrogen peroxide we went with a protocol involving nitrate, nitrite and NO. Secondly we wanted to measure lycopene. We adapted the protocol used by Cambridge 2009 iGEM as they had success and produced data, suggesting we would also be able to follow this.

The detection of Lycopene is simple since it has a distinctive red colour enabling it to be determined by spectrophotometry.

The protocols can be read here.

References

The Hallmarks of Aging. C. López-Otín, M. Blasco, L. Partridge, M.Serrano, G. Kroemer. Cell,153, 1194–1217, June 2013.

Comparative analysis of lycopene in oxidative stress. PD Sarkar, T Gupt, A. Sahu. Journal of the Association of Physicians of India, 60, 17-19, July 2012.