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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.

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.

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.

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.

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