The beginning of the laboratory work

MilliQ® or distilled water?

After we checked the results, we accorded to change different parameters. We changed medium distilled water to milliQ® water because we though that the little amount of chloroform present in distilled water might be able to activate gfp expression. Also, we suspected that chloroform concentrations in test tubes could be modified due to its volatility, so we tried to completely fill the falcons in order to minimize the chloroform losses. Finally, we represented normalized fluorescence versus chloroform concentration.

Kinetic studies with different incubation times and concentrations

The aim of these experiments was elucidate how the incubation in chloroform of the microorganisms used affects in the quantity of GFP produced by our biological system. The two main variables to control were the time and the amount of chloroform added. The first one is related with the fastness of the network of reactions which constitute our system, and the second one shows if the biosensors would be able to be used in a quantitative way as well (the principal objective of our project).

Inclusion bodies

A common limitation of recombinant protein production in bacteria is the formation of insoluble protein aggregates known as inclusion bodies. From a molecular point of view, inclusion bodies are considered to be formed by unspecific hydrophobic interactions between disorderly deposited polypeptides, and are observed as ‘molecular dust-balls’ in productive cells .The folding into a precise three-dimensional structure is a requisite for protein activity. However, under heat shock and other stresses protein folding can be impaired and folding intermediates then tend to associate through exposed hydrophobic patches. Individual polypeptide molecules are thus being trapped into growing oligomeric aggregates lacking the biological activity.

The solutions proposed and tested are: the addition of chloramphenicol in low doses in order to stop the synthesis of protein and then put the culture in an isotonic solution such as PBS. The other one is decreasing the incubation temperature to decrease the protein synthesis rate. Otherwise GFP will not have enough time to fold properly and it will probably form inclusion bodies.

The next question to answer was: How long will it take to protein to fold properly? ...in other words…How long is it necessary to incubate in PBS? The usual time was 1 hour but a kinetic study in PBS was done. We took samples every 20 minutes after putting the culture in this medium and the fluorescence and DO600nm were measured. Theoretically, whether inclusion bodies are formed, the fluorescence will increase after some time.

PCR perform

We finally got ready to perform a PCR (Polymerase Chain Reaction) to amplify amo, mbla and clpb sequences. We took a sample of each PCR tube and did an electrophoresis to see how separate the DNA bands were, but the results were negative. We suspected that were due to an insufficient DNA mould, so we put some Nitrosomonas cultures in order to obtain more biomass. This would take approximately 10 days, and while they grew we tried a new experiment based on the following hypothesis taken from some scientific papers: bacteria may need at least 12 hours to detect the presence of chloroform. So that we started a new 12-hour-kinetik assay, measuring the fluorescence every 2 hours and a half until 12 hours. Chloroform concentrations of 30, 300 and 3000 µM were assayed.

MilliQ® or distilled water?

Distilled water has a small amount of chloroform, and we thought it could be able to activate gfp expression. We made parallel experiments with both milliQ® and distilled water in order to discover if that amount could affect our research. So we made mediums and PBS solutions with those kind of water.

Finally, results showed us that the difference in chloroform between the two types of water produced no observable variation in gfp expression

PBS

Previous studies proved that sample incubation in PBS solution may amplify the fluorescence signal when working with GFP protein.

However, when we were making our fluorescence measurement, we noticed that it was lower in samples which were incubated less. Then, we decided to do a kinetic experiment about how PBS affected our samples.

The results showed that there was a top fluorescence at 30 minutes, but after this maximum, fluorescence declined with time. Thus the optimum time for incubation were 30 minutes.

It is due to the small amount of GFP in our samples. If we had used GFP-full samples, the optimum time would be higher.

Volatilization

When we performed the assays, one of the problems we had to think about was related to chloroform volatility. It was reasonable to suppose that chloroform concentrations could be modified due to that factor. Consequently, we tried to avoid chloroform losses by completely filling up test tubes, but it was useless.

After that, a gas chromatography was done to check that fact; nevertheless, there was no appreciable variation in chloroform levels with time. This is a logic result because all chloroform concentrations we tested were under its maximun water solubility. All of these conclusions can be contrasted in the following graphic.

Induction experiment

For the induction experiment we followed a series of steps:

• The day before the tests we inoculated a LB medium with our bacteria.

• The day of the tests:

o We pelleted the culture and resuspended in new LB medium.

o We added the chloroform in several concentrations, from 0 to 3000μL (0, 7, 33.5, 84, 100, 168, 300, 1000, 1500 and 3000 μL). The standard test used 0, 33.5, 84 and 100 μL.

o We incubated the aliquots in different times, from 30 minutes to 120 minutes. The standar test used 30 and 90 minutes.

o We pelleted again and resuspended in PBS.

o We incubated 30 minutes in PBS.

o We measured fluorescence.

The highest concentrations turned out toxic to our cultures. This is reasonable considering they were higher than those which there are in drinking water.

The tests were run with three strains, a DH5alpha E.coli, a mbla promoter-mutant E.coli and a clpb promoter-mutant E.coli. But results showed an increase in fluorescence with time in all the strains, thus there were no conclusive conclusions.

AMO

Our project was based on the ability of gfp-recombinant Nitrosomonas europaea strain to detect and metabolize chloroform, and thus express GFP. It’s supposed that N.europaea is able to do that due to the presence AMO, which is believed to recognize unspecifically this pollutant and oxidizes it to phosgene. Then, gfp expression would be activated.

So that, primer sequences were dessigned in order to clone amo gene by PCR. Nevertheless, after performing the PCR, purification and electrophoresis, we could observe the absence of the expected DNA.

It was obvious that something was wrong, and it was thought that it could be due to the small amount of “mould” DNA. Therefore a N.europaea strain was grown up, but it took us about two weeks. When whe had a suitable optical density, we proceed to repeat the PCR. Unfortunately, we reached the same results. This conducted us to conclude that there was a mistake designing primer sequences.

So, we could not obtain an amo-recombinant E.coli strain, and thus we could not reach our goal.

Inclusion bodies

As it was mentioned above, we tried to add chloramphenicol in order to inhibit protein synthesis and avoid producing those inclusion bodies. However, we didn't obtain any evidence about this; even more, it seemed that chloramphenicol contributed to slightly decrease fluorescence signal.

After that, we could check, by carrying out confocal microscopy, that there is not any inclusion bodies production. Thus, this fact could not affect neither gfp concentrations nor signal strength in our assays.