Lycopene Probiotic

Lycopene is an antioxidant naturally occuring in tomatoes, giving them their rich red colour. This antioxidant is also known to be lacking in the elderly population, meaning there are higher levels of unregulated oxidative stress. This BioBrick is induced by oxidative stress and produces lycopene as means to 'mop up' the oxidative stress in the environment, in the form of a probiotic.

OXIDATIVE STRESS

Early in our discussion of what we could target in ageing, we spoke to various academics, 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.

We have been researching the link between protein aggregation and oxidative stress. By using Dynamic Light Scattering (DLS), we were able to see that the size of a protein (monoclonal antibody) changed in the presence of oxidative stress. Under oxidative stress the protein becomes unstable and either undergoes a conformational change forming an unstable intermediate with a radius of a bit more than 1nm (as seen in image 2), or it aggregates (seen as the 2nd peak (at ~100-1000nm of the 2nd picture). 5mM of Hydrogen Peroxide was used to induce oxidative stress.

Particle Size Distribution without Oxidative Stress

 

Particle Size Distribution with Oxidative Stress

 

We also did some experimentation using Self-Interaction Chromatography (SIC) (data not shown). Without oxidative stress we get a nice peak and the area under the peak can be used to calculate the 2nd Osmotic Virial Coefficient (B2). B2 is a dimensionless no. that indicates protein stability. A positive B2 means that the proteins particles repel each other and will tend not to aggregate, but a negative B2 means that the protein is unstable, as its particles have a net attractive interaction and will aggregate. Without oxidative stress, the protein is stable with a B2 of 0.66.

However, when the same protein is exposed to oxidative stress we have a negative B2 value of -21 meaning that the protein is very unstable. This is because oxidative stress causes particle-particle interaction to become very high and very attractive. This means that the particles begin to take more and more time interacting with each other and the column attracts the mAb and heavily disrupts the flow of it.

In conclusion, oxidative stress has a very significant impact on protein stability. As you age and there is more oxidative stress in the body, the proteins in our cells which are essential to healthy functioning become unstable/aggregate, thereby leading to a loss of activity. This loss of activity of the essential proteins leads to the deterioration of health, so targeting oxidative stress to promote healthy ageing can be an effective strategy.

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

Our lycopene probiotic will act in the gut to neutralise ROS to regain the balance as shown in the diagram above

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.

 

Chemical structure of lycopene

We are looking towards using this as a probiotic treatment which also enables us to make the use of Dundee Schools gut simulation to test the workings of this.

Proposed transformation for probiotic in a safe chassis