Team:Valencia Biocampus/ResultsUpdated

From 2013.igem.org

Revision as of 20:39, 28 October 2013 by TonnyESP (Talk | contribs)

Show/hide wiki menu

The Riding

In this synthetic symbiosis, C. elegans acts as a transport for engineered bacteria (Pseudomonas putida) in order to take them to the hotspot of interest, because bacteria are not able to move fast through solid or semi-solid substrates but they are very interesting from a biotechnological point of view. This is the reason why we thought about this innovative mode of transport: the regulated formation of a biofilm.

To achieve our goal, we constructed a BioBrick (see part: BBa_K1112001) consisting of the coding sequence of the hmsHFRS operon, an adhesion operon natural from Xenorhadbus nematophila which allows the formation of a biofilm on the nematode S. carpocapsae, under the control of a nitrogen sensitive promoter (pGlnA, characterized by the 2012 Valencia Biocampus iGEM team). (Fig.1)

Controlling the mechanism

Riding is a regulated process: with low nitrogen in the media, the promoter is activated and hms genes are expressed, triggering the formation of the biofilm over C. elegans; in contrast, with high nitrogen concentrations, such as the ones found in nutrient-rich hotspots, the promoter is repressed, so bacteria can “get off” of the nematode (Fig. 2).


The manufacturing of the plates can be seen in this video.

Biofilm formation in genetically-engineered bacteria

Our original idea was to introduce the Biobrick in Pseudomonas putida , a bacterial species with wide applications in biotechnology. To do so, we cloned the construction (Fig.1) in the pIZ1016 vector, which has a replication origin compatible with Pseudomonas. We successfully performed the cloning (Fig.3), but the efficiency of the transformation was too low, so we have not been able to obtain P. putida transformants as of yet. This is probably a consequence of the length of the construction, 6,5 kb, which decreases transformation efficiency.

But far from being disheartened, we decided to express the construction with E. coli. We cloned the construction in the pUC57 vector, obtained transformant E. coli, and then grew them in medium with low nitrogen (0.6 g/L) in order to induce the formation of the biofilm. C. elegans was fed with these induced bacteria, and then several worms were isolated in order to check biofilm formation with scanning electron microscopy (SEM) imaging. As you can see in Fig.4, it actually worked! We observed a formation of an E. coli biofilm over the nematode!

The Calling

Looking for an attractant

When we were considering setting up a transport for bacteria, we thought it was necessary to have a 'destination', a place to go. That destination would be a 'hot spot' on a heterogeneous substrate where Caenorhabditis elegans should carry the bacteria to.
Leveraging the powerful smell of the nematode, it was decided to try a number of attractants from various lists from the web www.wormbook.org that could work as 'hot spots' in our experiment. Thus, using C. elegans chemotaxis, we would be able to direct transport.

The attractants experiment

The test was carried out by creating our own plates in which half plate would be unmodified NGM and the other half would include soluble compounds before solidifying, or after solidification (in the case of volatiles). The list of modifications can be found in Fig. 1.

Table 1

To place C. elegans on the plate, different cuts were made on a fresh plate of NGM and the resulting small pieces were placed in the exact center of the 50% - 50% plates to determine which side of the plate the nematode preferred, one per Petri plate.

The results after counting 2 replicates per attractant can be seen in Fig. 2.

Table 2

From Table 2 many of the attractants that were thought viable were discarded.

Volatile attractants were not a good choice because they evaporate quickly (which would also be a limitation for the field experiments).

Another impediment arose. Once we had already performed the experiments, we decided that the promoter that would control the production of interference RNA in Escherichia coli would be regulated by nitrogen, so amino acids had to be discarded as attractants; because it would modify the controlled expression of E.coli.

That left MgSO4 and hypoosmotic media as potential attractants.

Attractant final choice

Because the hyposmotic medium could interfere with the proper growth of bacteria (our nematode's food), the final decision was to choose MgSO4 as our attractant.

The question at that time was: How can we multiply the amount of MgSO4 to increase the efficiency?

MgSO4 efficiency

Once we had selected the most feasible attractant for the ‘transport’ during our experiments, we needed to know which could be the largest concentration of MgSO4, in order to optimize the nematode's attraction.
To choose the concentration, attraction was tested in a battery of increasing concentrations (regarding the initial medium of 1 ml / L). Bearing in mind the results of the x2 factor, we decided to test other factor concentrations: x3, x4, x5, x8 and x10; covering a wide range that did not exceed the concentration that would affect the life of C. elegans or bacteria.
Moreover, experiments with E. coli and Pseudomonas putida were carried out. These trials were performed in order to obtain the final media: half plate with non-altered NGM and half with the medium for PHA production by Pseudomonas and interference RNA production by E. coli.
Not knowing which fatty acid could activate the transcription process better, we tested two possibilities: oleic acid (named in the tables as PHAol) and octanoic acid (PHAoc). The concentration for each fatty acid was tested in E. coli and we obtained best results with:
  • 1.28 µl/ml of Octanoic acid.
  • 2.58 µl/ml of Oleic acid.

You can see our results in figures 3, 4, 5 and 6. The first two counts were made after 3 hours and the next ones after 6 hours. We supposed that after that time, the worms were already capable of selecting their "favourite" side of the plate, get to the desired side and keep moving in the same part of the petri dish. We prepared control plates with the same composition but without MgSO4 in PHA media.

Tables 3, 4, 5, 6

Final concentration choice

Once the tests were performed and after observing the results obtained with the different selected fatty acids, we then observed a scattered result(there was probably a repellent effect at high concentrations in the medium with oleic but reversed in the octanoic) and in the end, we decided to use the following MgSO4 concentrations for each medium:
  • If oleic acid is used: Best results with 4ml/L MgSO4.
  • If octanoic acid is used : Best results with 10 ml/L of MgSO4.
Octanoic was discarded as a transcriptional activator of the iRNA of clumping after other results for E. coli were obtained, so finally we selected the PHA medium with oleic acid only.
The biggest problem while trying to have an effective attractant was knowing how effective could an attractant, in the presence of E. coli, be. It was therefore necessary to test the tradeoff between the value of 4ml / L MgSO4 and pair it with different concentrations of bacteria, high enough to feed the nematode but low enough to allow the attractant effect of MgSO4.
To find this point of commitment we prepared experiments in which the concentration of 4ml/L of MgSO4 was faced against different ODs from serial dilutions of a preculture of E. coli DH5a.
Table 7 shows the results obtained. The count took place 3h after the passing of fresh nematodes.

Best E. coli OD choice

An OD of 1 (minimum concentration of cells / volume) gives an attractive effect even better than expected (subsequent experiments try to check if there is synergy between E. coli and attractive factors MgSO4).
To improve the approximation we decided to repeat the experiment with MgSO4 concentration and the chosen bacterial OD showing that the system worked correctly. The results can be seen in figure 8.

Table 7