Part:BBa_K1954001

This is UCL iGEM 2016's nomination for Best New Composite Part as well as its new BioBrick submission for a Silver Medal (Experimentally validate that at least one new BioBrick Part or Device of your own design and construction works as expected).

For UCL iGEM 2016's entry for the Gold Medal criterion, 'Improve the function OR characterization of a previously existing BioBrick Part or Device', please go to: BBa_K239009.

Lycopene cassette under the control of NarK, an oxidative stress inducible promoter

Lycopene expression under control of NarK promoter to enable cells to grow better under conditions of oxidative stress.

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 see our wiki page: http://2016.igem.org/Team:UCL/HP/Silver/Experts.

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.

This data is shown on our wiki page: http://2016.igem.org/Team:UCL/Gut Under "oxidative stress"

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.

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 stomach 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 Notes

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:

Sensing The promoter senses oxidative stress and in response turns on transcription of the downstream genes. Responding The gene for the antioxidant, lycopene is turned on as a response to oxidative stress.

Lycopene-NarK initial design:

Further steps were taken in the design process:

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.

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.

Characterisation for Silver Medal by UCL iGEM 2016

UCL iGEM 2016 created and characterised this biobrick for the Silver Medal.

mNARK-Lycopene Device Characterisation

mNARK lycopene enables ecoli growth under Hypoxia conditions

Hypoxia is a condition in which cells are deprived of oxygen due to low concentration of oxygen in the extracellular milieu. In humans, low oxygen levels in the blood affect tissues. Oxygen is essential for diverse cellular functions, such as catabolic and anabolic processes, and low intracellular concentrations have a negative impact on cell functions and survival.

Oxygen deficit can have a severe impact on cellular function, as seen in cell stress. The inability of cells to effectively manage cellular stress over time has been linked to cellular ageing and age-related diseases (Haigis and Yankner, 2010; Poljšak and Milisav, 2012). Cellular stress leads to deregulation of intracellular processes, as both the structure and function of macromolecules are compromised. Furthermore, high amounts of ROS have been implicated in cellular stress and ageing (Poljšak and Milisav, 2012).

We performed an additional assay expressing lycopene under the mNARK promoter, to test if the cells could survive longer under hypoxia-induced stress. E. coli cells transformed with this construct were compared with the wild type TOP10 E. coli (W/T) monitoring growth and division via optical density (OD) at 600 nm, at specific time points – 3 hours and 16 hours following withdrawal of oxygen.

mNARK-Lycopene cells had a higher OD compared to the wild type cells. Cells exposed to hypoxia were also compared with the cells that were grown with oxygen. Most cells still survived despite the presence of hypoxia.

Furthermore, with regards to W/T cells, a depletion in the oxygen concentration caused a drop in cell growth and division, as reflected in decreased OD measurements. However, growth and division of mNARK-Lyco-containing cells was maintained in oxygen-deficient environment during the 16-hour time test period.

This shows that our BioBrick construct (mNARK-Lyco) was able to ensure cell growth and division in oxygen-deficient environment.

The mNARK-Lycopene device 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.

Under various stresses, the mNARK-Lycopene device promotes E. coli growth. Characterisation was achieved by comparing the performance of the BioBrick against wild type E. coli.

Initially, the growth of mNARK-Lycopene was compared with the growth of wild type E. coli. The mNARK-Lycopene cells substantially outperformed the wild type cells, reaching a final OD600 of around 0.45 and, potentially, still rising, while the growth of the wild type cells had clearly levelled off over the equivalent time period and had began to die, reaching a final OD600 of around 0.12.

After carrying out the positive control, the mNARK-Lycopene cells were tested under conditions of simulated oxidative stress, with the LB media containing 2 mM copper (II) chloride. The results indicate that the mNARK-Lycopene device boots E. coli growth. Wild type E. coli grew to a final optical density of 0.35 after three hours, from an initial OD of 0.15. The mNARK-Lycopene progressed from an initial OD of 0.25 to a final OD of 0.60.

The BioBrick was then tested against simulated oxidative stress conditions of 50 and 100 µM sodium nitroprusside. Both of these experiments, again, indicate that, in the presence of simulate oxidative stress conditions, the mNARK-Lycopene promotes E. coli growth substantially better then the control. At 50 µM, the optical density of the wild type cells decreases to an OD of 0.05 from the initial reading of 0.17. In comparison, the mNARK-Lycopene cells achieved a final optical density of 0.33 from a low of 0.05. At the 100 µM concentration, the wild type cells flat lined, remaining at an optical density of 0.25, while the lycopene cell concentration increased substantially from an optical density of 0.07 to 0.45 after four hours of growth.

In summary, out analysis has shows that our mNARK-Lycopene device protects the cells from oxidative stress and, from this, we can assume that this effect will continue once our lycopene probiotic is consumed by and aging person. Additionally, it has been proved that the cells will be able to survive and multiply in the gut and colonise it. Therefore providing protection to neighbouring cells by ‘mopping up’ the oxidative stress.

The figure above shows that the mNARK-Lycopene Device promotes E. coli growth better under oxidative stress. The figure above shows that the mNARK-Lycopene device promotes E. coli growth better under oxidative stress.

After establishing that the mNARK-Lycopene device improves cells growth compared to wild type E. coli, the growth of the Lycopene cells was measured against lycopene expression. OD485 is indicative of Lycopene expression, as described by team Cambeidge iGEM 2009, whilst OD600 represents cell density. Any OD485 readings from the wild-type cell samples represent background noise, and the difference between the OD485 measurements for the mNARK-Lycopene cell samples and the wild-type cell samples can be accrued to Lycopene expression.

First, the cells were grown in LB media with 2 mM copper (II) sulphate. As shown in the plot, the mNARK-Lycopene device reached a higher cell density of 0.57, which corresponds to an OD485 of 0.68. Wild type E. coli does not achieve such a cell density and, by extension, lycopene expression. From an initial cell density of 0.08, the wild type cells reach a maximum cell density of 0.34. Additionally, the gradient of the graph represents the rate at which cells express lycopene. The plot indicates that the mNARK-Lycopene device expresses lycopene at a higher rate than the wild type cells. It is clear that, for both the mNARK-Lycopene device and wild type E. coli cells, lycopene expression increases with cell density, as more lycopene is being produced as a result of there being a larger number of lycopene producing cells. Therefore, it can be concluded that the mNARK-Lycopene mops up the oxidative stress, which prevents the cells from dying. Therefore allowing cells to grow to a greater optical density, and, as mentioned above, greater cell density corresponds with greater lycopene production.

The second graph is derived from experiments using LB with two different concentrations of sodium nitroprusside (SNP): 50 μM and 100 μM. During the experiment with 2 mM copper (II) sulphate; the wild type E. coli cells continue to grow, since it did not produce an environment where there was substantial oxidative stress. Sodium nitroprusside can create an environment with greater oxidative stress. Due to the greater oxidative stress, the wild type cells are unable to grow in these conditions and show a very low optical density at 485 nm, which indicates a lack of lycopene production. The gradient of the two wild type cell graphs are similar, which is expected, as neither have an oxidative stress promoter. Wild type cells grew better under less oxidative stress (50 μM SNP), compared to 100 μM SNP. The optical density at 485 nm is lower for higher oxidative stress conditions with the wild type cells, which is due to their respective cell densities. However, the mNARK-Lycopene cells continue to grow and produce lycopene in these conditions. The lycopene cells display greater growth in the harsher 100 μM conditions compared with 50 μM. The gradient of 50 μM mNARK-Lycopene at 0.8435 is less than that of the 100 μM sample, which is 1.0864. This indicates that our mNARK device increases lycopene expression under higher oxidative stress. In this experiment, the performance of the Lycopene cells is more pronounced, clearly showing that the mNARK-Lycopene devlice mops up the oxidative stress, which prevents the cells from dying. This results in a greater optical density being achieved and, by extension, greater lycopene production.

From the data below, we can deduce that our biobrick will produce more lycopene when there is more oxidative stress in older people there creating a regulatory feedback system.

Hence, we have characterised our silver BioBrick.

The figure above shows that the mNARK-Lycopene device promotes E. coli growth better under oxidative stress.

The figure above shows that the mNARK-Lycopene device promotes E. coli growth better under oxidative stress.

===Demonstration and Collaboration: Stomach Simulation === Following our talks with Aubrey de Grey and Filipe , we really wanted to conduct experiments that show that our lycopene probiotic can survive in the gut and acidic conditions of the stomach. We were very lucky to have met with Dundee GEM team at the UK GEM meet up where they told us about a stomach simulating device that they own. The bacteria in a sealed container and reconstructing the conditions found in the stomach. A manual pulley system replicates the churning of the stomach with an acidic environment. You can read more about team Dundee 2016's ACME Stomach Masher here: http://2016.igem.org/Team:Dundee/Proof

We sent out a slab of bacteria that contained our lycopene biobrick within it, to test whether our bacteria could survive in the stomach, in order to provide proof that our probiotic idea would be functional in the human body. Dundee then measured the growth of our bacteria by taking optical density measurements every 15 minutes for an hour since adding it to their device. The results were extremely promising; our genetically modified bacteria grew successfully in their device.

This graph shows that OD, and hence E. coli growth can occur in the stomach.

This graph shows that lycopene indeed can be detected as shown by measuring absorbance at 400nm.

Thus, we have characterized this biobrick. This is directly as a product of the integration of our human practices into the design. Beyond this we have demonstrated that it works in simulated real-life conditions through a wonderful collaboration with Dundee Schools, thereby illustrating how this one biobrick embodies the spirit of iGEM.