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

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:

 

 

Simplified diagram of our lycopene BioBrick. 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 500 bp 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.

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.

RESULTS

THE AGEING GUT

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.

INTEGRATED HUMAN PRACTICES

After choosing our topic of healthy ageing we spoke to various researchers in the area. We especially valued the opportunity to speak to Aubrey De Grey, an author of books such as 'advocate for an indefinite human lifespan' and 'ending ageing'. He is the co-founder of SENS research and is renowned in the field for his research. We were particularly looking forward to discussing the idea of a antioxidant probiotic with him following his interest in the free radical theory of ageing and his positive view that "Ageing is emphatically not an inescapable destiny".

In conversation with Aubrey de grey, it was suggested to us that our lycopene probiotic would need to be controlled. This is because reactive oxide species are useful in important signalling processes which act to protect the cell. Taking on board this advice, we considered a method to control lycopene expression.

 

 

To achieve this we decided to look through the registry at existing promoter which would select lycopene expression only in 'high stress' conditions which mimic the imbalance of stress within the cell. Hence, we decided to combine lycopene with a stress sensitive promoter which would only release lycopene when cells are stressed. This promoter was NarK. We are very grateful to Aubrey for his insight and enthusiasm for our project, and also thankful that he took time to speak with our team and influence our project in a positive way.

To watch our conversation click here.

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

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.

Beyond this the combination of lycopene with our NarK promoter, the release of lycopene is controlled specifically in response to oxidative stress. This has an advantage over lycopene within food which is all released all at once during digestion and may in fact result in the body containing more lycopene than it can process, meaning more energy would need to be expended to digest it.

Further still probiotics are a more long term solution, as it has been shown that E. coli can occupy the gut for even 3 months! This is a huge advantage as it would not contribute to existing daily medication the elderly population are often burdened with. It also doesn't rely on a diet that is rich in lycopene, which given the price and availability of these fruits, especially watermelon, could in the long term be a more economical solution.

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:

 

 

Simplified diagram of our lycopene BioBrick. 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 500 bp 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.

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.

RESULTS

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.