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Team:Valencia Biocampus/Project
From 2013.igem.org
Project Overview
Escherichia coli
Hola a todos!
Pseudomonas putida and PHA production
Pseudomonas putida is a gram-negative bacterium that is found in most soil and water habitats where there is oxygen. Its diverse metabolism and its capacity to break down organic harmful solvents (it has most genes involved in degrading aromatic or aliphatic hydrocarbons) in contaminated soils make this microorganism irreplaceable for research studies in the field of bioremediation but also for biosynthesis of value-added products. In addition, Pseudomonas putida has several strains including KT2440, the one we have worked with. This strain can colonize plant roots, from which they take nutrients, while at the same time it offers protection to the plant from pathogens.
For example, it is capable of converting styrene oil into the biodegradable plastic PHA. This helps to degrade the polystyrene foam which
was thought to be non-biodegradable. Styrene is a major environment toxic pollutant released from industrial sites. The conversion to PHA allows the cure
of styrene pollution but it is also beneficial to society because of its applications in tissue engineering.
PHA is also environmental friendly and has a long self-life therefore it is also used in everyday items. Unlike styrene, PHA can break down in soil or water.
Within Pseudomonas putida, PHA accumulates under unbalanced growth conditions as a means of intracellular storage, storing excess carbon and energy. These PHA polymers are synthesized by enzyme PHA synthase which is bound to the surface of the PHA granules and uses coenzyme A thioesters of hydroxyalkanoic acids as substrates.
The role of P. putida in the Synthetic symbiosis that we have designed is to be carried by C. elegans to hotspots of interest where would produce bioplastic PHA.
An overview about our nematode
One of the main characters of our work is a nematode known as Caenorhabditis elegans, from the Rhabditidae family. It was first used as an experimental model in Developmental Genetics studies and nowadays is also used in other fields such as Clinical Biology, Neurobiology and Cell Biology, being a good model to study Alzheimer disease, obesity, diabetes and aging, among others.
Another interesting thing is that it feeds on Escherichia coli. Its “favorite” strain is OP50, although we checked that it’s also able to feed on XL1-Blue strain, the one that we used in all our molecular biology experiments.
Some advantages of C. elegans when compared with other model organisms are:
- Lifespan ranges between 2 and 3 weeks, so experimentation times are reduced.
- Its maintenance and study is cheap and simple (it is transparent, which facilitates microscopic observation).
- It is very small (1 mm), so it is possible to carry out experiments with a huge number of worms in a small Petri dish having a great statistical support.
Why use C. elegans as a transport?
When we were considering on creating a new system for the transport of bacteria, we found different key advantages that made the nematode the best option. For example, C. elegans is able to move very fast around solid substrates (soil is, actually, its natural habitat) and also in agar, so it’s very useful for both lab experiments and real-environment tests.
Its movement, in addition to being very fast, has two modes: random and directed.
When there is no attractant in the medium, C. elegans moves doing uncoordinated movements in several directions in what is known as 'random walk'.
The situation changes when there is an attractant in the medium. Here, our 'transport' begins to direct his movements to the focus of the substance (volatile or soluble) which acts as an attractant in a process known as 'chemotaxis' thanks to the amazing smell of our nematode. This allows us to ‘guide’ the nematodes towards defined spots in irregular substrates.
Chemotaxis is the foundation to guide the transport of bacteria and is therefore the focus of experimentation with C. elegans, with the aim of finding the best attractant. This ability makes C. elegans a perfect 'bus' for bacteria. (simuelegans online here)
Moreover, we found two pathogens (Yersinia pestis and Xhenorhabus nematophila) with the ability to form biofilms on C. elegans thanks to the proteins of the operon hmsHFRS. When genetically-engineered strains of commonly used bacteria such as Escherichia coli or Pseudomonas putida express this hmsHFRS operon, they have the 'ticket' to travel: they are able to adhere to the worm’s surface by means of forming a synthetic biofilm.
Expanding our knowledge about C. elegans...
Once we knew that C. elegans was the best option, we began to discover interesting things for further study.
There are several strains of our nematode. The one that drew our attention was called 'N2', which had a fairly interesting behavior: under normal conditions, it eats individually, whereas under certain conditions (such as starvation), a social feeding behavior known as “clumping” is induced. But this phenomenon can also be induced if the expression of some particular genes is interfered. This fact gave us the opportunity to develop the first artificial symbiosis between worms and bacteria, based on the manipulation of the behaviour of C. elegans by simply nourishing it with transformed E.coli able to synthesize the iRNA.
With it, while C. elegans acts as transport, bacteria return the favour giving it the ability to eat in company.
Results
The Riding
The Calling
The Calling
Looking for an attractant
When we were considering setting up a transport of bacteria was thought to be necessary to have a 'destination', a place to go. That destiny would be a 'hot spot' on a heterogeneous substrate where Caenorhabditis elegans should lead right to that point the bacteria.Leveraging the powerful smell of the nematode, it was decided to try a number of attractants from various lists from web www.wormbook.org that could work as 'hot spot' of our experiment. Thus, using the C. elegans chemotaxis, we could direct transport.
The attractants experiment
The test would be carried creating our own plates on which half would be NGM unmodified and the other half part would be including soluble compounds before solidifying or after solidification in the case of volatiles. The list of modifications can be found in Fig. 1.
To place C. elegans on the plate, different cuts were made on a fresh plate of NGM, the resulting small pieces were placed in the exact center of the 50% -50% plates to determine which side of the nematode preferred, one per Petri plate.
The results after counting 2 replicates per attractant can be seen in Fig. 2.
From Table 2 it could rule out many of the attractants that were thought viable.
Volatile attractants were not a good choice to evaporate quickly (which is also limited to the field experiments).
Another impediment arose. Once had already performed the experiments, it was decided that the promoter that will control the production of RNA interference in Escherichia coli would be controlled by nitrogen, amino acids had to be discard as attractants; it would modify controlled expression of E.coli.
That left the MgSO4 and hypoosmotic media as potential attractants.
Attractant final choice
Because the hyposmotic medium could interfere with the proper growth of bacteria (food of our nematode), the final decision was to choose the MgSO4 as attractant.The question that arose at that time was: How we can multiply the amount of MgSO4 to increase efficiency?
MgSO4 efficiency
Once we had selected the most feasible attractant to the ‘transport’ for our experiments, we needed to know what could be the largest concentration of MgSO 4 in order to optimize the attraction of the nematode.To choose the concentration, were tested in a battery of increased concentrations regarding the initial medium (1 ml / L). Bearing in mind the results of the factor x2, we decided to test other factor concentrations: x3, x4, x5, x8 and x10; covering a range that does not exceed the concentration at which might affect the life of C. elegans or bacteria.
Moreover, approaching experiments with E. coli and Pseudomonas putida, these trials were testing the end media: half plate with NGM non-altered and half as PHA production medium for Pseudomonas and interference of E. coli.
Not knowing what fatty acid could activate transcription 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 choosing as better:
- 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 two first counts were made after 3 hours and the next ones after 6 hours. We suppose at that time the worms can select their “favorite” half part gone across and their movement would be always in the same area. We prepare as control plates with the same composition but without MgSO4 in PHA media.
Final concentration choice
Once the test already performed and that the results seen by the factor of PHA and MgSO4 selected fatty acid give very scattered (there is probably repellent effect at high concentrations in the medium with oleic but reversed in octanoic) we make a selection of MgSO4 concentration for each medium.- If it is used oleic acid à Better results with 4ml/L MgSO4.
- If octanoic acid used à Best results to 10 ml/L of MgSO4.
The biggest problem in trying to have an effective attractant was how effective could be in presence of E. coli. 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 permit the attractive effect of MgSO4.
To find this point of commitment we prepare experiments in which the concentration of 4ml/L of MgSO4 is faced against different ODs from serial dilutions of a preculture of E. coli DH5a.
Table 7 shows the results. The count took place at 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 see if there is synergy between E. coli and attractive factors MgSO4).To improve the approximation was decided to repeat the experiment with MgSO4 concentration and the chosen bacteria OD showing that the system works well. The results can be seen in figure 8.
The Clumping
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The Building
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Parts
These are the BioBricks we have designed, constructed, and characterized. We have submitted them to the Registry of Standard Biological Parts
| Works? | Name | Type | Description | Designer | Length | Structure |
|---|---|---|---|---|---|---|
 |
BBa_K1112000 | Regulatory | fadB promoter + FLP-21 iRNA | Pedro Luis Dorado Morales | ||
|
BBa_K1112001 | Regulatory | pGlnA + hmsHFRS operon | Alba Iglesias Vilches | ||
|
BBa_K1112002 | DNA | Cluster PHA | Alba Iglesias Vilches |
BBa_K1112000: fadB promoter + FLP-21 iRNA
This construction is made up by a fatty acid-sensitive promoter that directs the transcription of an iRNA responsible for the social or solitary behavior of C.elegans.
BBa_K1112001: pGlnA + hmsHFRS operon
We reused our nitrogen sensitive promoter from one of our last year constructs and we employ it to control the expression on the operon that triggers the formation of a biofilm over C.elegans.
BBa_K1112002: cluster PHA
This BioBrick contains the complete natural sequence that induces the production of PHA in Pseudomonas putida (KT2440).
