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Team:Evry/Biology/Chassis
From 2014.igem.org
Biology - From the unknown depths of the ocean to a chassis in iGEM
A specific chassis for specific environments: Seas & Oceans
== A duo from the sea ==
As seen in the overview our biosensors should work optimally in native marine conditions (approximately 24-30 g/L NaCl). Moreover it has to attach to sponges and stick with them without disturbing the microbiome for as long as possible. Therefore it is necessary to use a bacterium naturally present on sponges, may it be an epibiont or a symbiont.
Unfortunately it is a condition for which no current chassis in iGEM is ready for. It is still worth noticing that an interesting first effort toward a marine chassis was initiated by UCL 2012.
To be up to the task the bacterium should:
- be massively present on the sponge surface / avoiding being in an unfavourable position for food competition.
- be found mainly in sponges / avoid spreading to species in contact with sponges.
- be the phenotypically closest possible to a known bacterium / avoid cell cultures difficulties.
To be up to the task the sponge should:
- be numerously present in the ocean / avoiding putting species in danger.
- be a natural host of the bacterium / avoid adapting its microbiome to the new epibiont.
- be easily grown in a laboratory / avoid sponge culture difficulties.
The closest combination sponge/bacterium that could fit these requirements is Spongia Officinalis / Pseudovibrio denitrificans
== Born to filter: Spongia officinalis ==
Spongia officinalis has been used for millennia and for various purposes: from the Greeks for bathing and lining their armours, to today's pharmaceutical attempts to produce anti-inflammatory and other therapeutics, not even mentioning the use by Arabic physicians as early as 932 A.D of soaked sponges with narcotic drugs to place over the patient’s nose to provide a state of anesthesia. In a nutshell everyone knows the "bath sponge".
Spongia officinalis is a leuconoid sponge. A leuconoid sponge has a thick body wall, and the ostia open into incurrent canals that draw water into the sponge’s body. These incurrent canals open into chambers that are lined with choanocytes. Water flows from these chambers into excurrent canals that empty into a relatively small spongocoel. From there, water exits through an osculum.
Unlike asconoid and syconoid sponges, which are basically built around their spongocoels and oscula, a leuconoid sponge has a complex, irregularly-shaped body that may have several oscula. It allows a leuconoid sponge to grow to larger size.
It also provides them with powerful filtration capacity (approximately 1200 times its volume per day) which makes it a perfect host for our system. But as mentioned earlier our aim is to engineer an epibiont and not the sponge itself.
Following this introduction, we will both describe what Pseudovibrio denitrificans exactly is and how we turned it into a transformable/selectable-ready chassis.
Pseudovibrio denitrificans the bacterium to modify
== From a poorly known genius, a jewel emerged ==
At first glance, working on the microbiome of sponges appears like a hazardous task: 5734 articles for marine sponges, only 51 with microbiome of sponges, only 28 articles mentioning Pseudovibrio genius, only 5 mentioning Pseudovibrio denitrificans, and finally none about a genetic engineering system in it.
Moreover with only 12 species in the genius, and 2 strains sequenced (Pseudovibrio sp. FO-BEG1, Pseudovibrio sp. JE062) we knew we would have to sequence our strain of Pseudovibrio denitrificans with a mapping to one of the 2 strains references. A formidable drawback even considering that in the first place the species was chosen because of its natural denitrification ability (see toxic compound) and easy to source at DMSZ.
On the other hand Pseudovibrio denitrificans, or strains related to the species, has been shown to be in the majority in at least six microbiomes of sponges in the Mediterranean Sea, where spongia officinalis resides.
Now the thorny question is: can it be easily used in our lab?
== A bacterium we could work with ==
In the literature Pseudovibrio denitrificans is described as a Gram-negative, motile by means of one to several lateral or subpolar flagella requiring NaCl for growth. It exhibits optimal growth at about 30 C°, pH 8 and 3 % NaCl, and a doubling time of 45 min in rich media. In a nutshell the conditions seem favourable for an iGEM project.
They are known to be capable of anaerobic growth by carrying out denitrifying metabolism using nitrate, nitrite or nitrous oxide as terminal electron acceptors. Being consequently a main reason for our RNAseq study to discover the transcripts upregulated by the presence of nitrite and clone their promoters as new sensors.
The following section describes protocols used to optimize (Rich media) & control (Minimal media) growth conditions.
