Team:USP-EEL-Brazil/Experiments
Cell Resistance
Our experiments should start with parameters, right? So our first stop here is to determine at which point would the fatty acids concentration in the medium would start to inhibit the cell growth and be actually prejudicial to the E.coli.
As tocopherol in high concentrations is also prejudicial to the cells, we should also quantify at which concentration it would become toxic, instead of beneficial.
Procedure
The first step to our experiment is to prepare the stock solutions and mediums, so we did, as following:
-Tocopherol was diluted in ethanol, to a final concentration of 10mM.
-At low concentrations, oleic acid was applied directly to the medium. In higher concentrations, it was first diluted in the reason of 2:1, oleic acid:ethanol.
-Optimum medium was prepared.
-Samples were made in duplicate.
-All measures were made in a plate reader, in triplicates.
Source: Personal archive.
Experiment
E.coli cells were inoculated in optimum medium and left to grow overnight in a shaker in 37º Celsius and 190RPM.
Based on Okai, et.al, we defined our initial tocopherol and oleic acid concentrations. In his experiment, he used free nonylphenols, which are actively toxic to organisms, so we already expected the oil concentrations to be higher than these first goals set. The growth medium was prepared using the optimum medium in falcon tubes, in 5mL samples.
Source: Personal archive.
Tocopherol quantities were added from the 10mM solution and added to the medium as follows: 0uL, 5uL, 25uL, 50uL, 100uL and 250uL.
The overnight grown cells were than inoculated into the medium with the tocopherol and placed in the shaker to grow. Measures were made until we reached 12h from our starting point.
As it is quite clear from the graphic, the 0.11M sample has been really capped by the tocopherol. Not only it inhibited the cell growth but it started out with a negative growth, until the cells stabilized themselves and grew a little. This concentration we then set as our far maximum concentration for tocopherol presence in the growth medium.
With the intent of confirmation that a single result was not a fluke and that tocopherol is indeed toxic in higher concentrations, we over exaggerated a few values as it follows: 250uL, 300uL, 400uL, 500uL and 1000uL. These values are transcribed into mol/L in the graphics.
This time the situation is much more critic. All relative cell growth were absolutely negative. In this trial, the 0.11M test did not end up growing, like the previous one. This can be considered normal, having in mind that both tests showed starting negative growth, but the latter one probably was not able to deal with the elevated concentration as well and ended up having a final negative growth.
It is also noteworthy that all curves have a similar pattern. It starts with a huge negative growth, then, the cells seem to start growing until they stumble and fall again. All curves, even if very small, had a resistance glimpse in the hours 3 and 4. This might indicate the cell's ability to deal with the exogenous susbstance, but inevitably failing to do so. This first graphic may also show a success of this theory, by having the 250uL sample to end up growing.
Just like tocopherol we needed to determine viable concentrations of oleic acid to be added into the medium, so we started with that quantities we found on Okai, et.al., which were relatively small and would easily dissolve in our medium, so were directly added. They were: 0uL, 0.5uL, 1uL, 1.5uL, 2uL and 3uL.
The graphic shows very similar relative cell growth and does not have any specific order for the relations of concentration to higher curves. The only exception noted is the 0.00032M sample, whereas it had a significantly increased growth rate. We can also note the control sample being the one with the lowest growth rate. This reflects the usage of the oleic acid by the cell's metabolism, therefore, the samples with the presence of oil had a bigger grow rate, taking to account that the cells could have spared energy needed for the oil pathway activation and instead, got it ready to use. It probably also mean that 0.00032M, is probably near the optimal point of oil concentration to stimulate cell growth.
So now, instead of stimulating, we wanted to find out the point in which the rich fatty acid medium would start being toxic for the cells. This meant having higher concentrations of oleic acid. We added in the following quantities: 25uL, 50uL, 100uL, 200uL, 400uL and 1000uL.
Since our medium is also rich in nutrients and phosphates, the addition of higher concentrations of oil would create a biphasic system inside the test tube, some nutrients and composts would go to the oily phase and the solubles would stay in the aqueous phase. The oil now rich in nutrients would also assume a more viscous state and a good chunk of it would stay "glued" to the tube walls when staying in the shaker.
The results until the 0.256M oil concentration in the medium presented satisfactory results, but the 0.514M and 1.28M samples were overloaded with the gooey matter and presented a huge distortion in the turbidity and consequently in the reading.
As we can see, even tough the cells did have a considerable growth, if compared to the results with lower oil concentrations, the later growth rates are clearly smaller, as the 0.128M of oil is the highest one and it still was outgrown by the control. We believe that the downfall of the 0.256M and 0.064 samples are not directly related to the oleic acid concentration, even tough they did probably contributed to it. It is also very reasonable about the 6h point of the 0.256M sample and affirm that it represents an experimental error and that the value shown in the graphic does not effectively represent the true value./p>
The graphic showing the 0.514 and 1.28uL samples are extensively high. These samples were really turbid and had the white concentrated oil with nutrients, which turned out really white. This meant that as we took samples for measurements, the more viscous and turbid the sample became and higher was the reading, meaning it would not reflect the true cell growth.
Genetic manipulation.
The iGEM kit arrived on June and when we first laid our hands on it, we had no experience with synthetic biology at all… We knew the theory needed to work with it, but when you step inside the lab, there’s a whole new world to explore. A whole new challenge to tackle.
iGEM kit plates. Source: Personal archive.
The constructs design created many discussions and not many methods available to us to do it. Initially we thought about synthesizing the genes all at once, resulting in two operons, one for Lux and the other for the tocopherol. But we then discovered that we had a limit of 2000bp per gBlock we could order. Our choice ended up being to order the genes separately and add them together with Gibson Assembly, this way we could deposit the parts one by one and the whole construct all together.
For the Gibson Assembly, we designed the genes to have around 30bp of overlap at each end of the parts, and the BioBrick prefix and suffix at the beginning and at the end of the operons' border genes. Each part has its own RBS before the gene itself. We also designed the primers, some with and some without the prefix and suffix. The idea was to make PCRs of the plain genes and amplify them for the Gibson. The ones with prefix and suffix would be directly deposited in the Registry.
Unfortunately, every experiment is not a walk in the park. Our assemblies would not work, and we couldn't pinpoint the problem. The products would show some incomprehensible bands. After much revising, we still can't understand why the Gibson Assembly did not work properly.
Gibson Assembly electrophoresis. Top half samples: Lux Operon. Bottom half samples: Tocopherol Operon. Source: Personal archive.
Some assembly variations were applied to different samples, to no avail. Although the composite parts were giving us a hard time, all the other iGEM protocols, including digestion, 3A assembly and competent cells worked neatly and pretty effectively.
Sources
Okai, Y.; et.al. Protective effects of alpha-tocopherol and beta-carotene on para-nonylphenol-induced inhibition of cell growth, cellular respiration and glucose-induced proton extrusion of bacteria (2000).
ALBERMANN, C. et al. Biosynthesis of the vitamin E compound delta-tocotrienol in recombinant Escherichia coli cells. ChemBioChem, v.9, n.15, p.2524-2533, 2008.
Washington iGEM. Make it or break it. Avaible at:https://2011.igem.org/Team:Washington
Hino, T.; Andoh, N.; Ohgi, H. Effects of beta-carotene and alpha-tocopherol on Rumen bacteria in the utilization of long-chain fatty acids and cellulose (1993)
