Team:UNIFI/Theoretical basis
Biological principles
Bacterial communication
Bacteria are unicellular organisms, but many of their biological activities are strictly dependent on the number of cells that are present in the colony. In fact, bacteria have developed some sorts of communication, based on cell-to-cell signaling and response, which allows them to activate specific functions only when the colony is big enough. This phenomenon is known as “quorum sensing”: the mediator is a self-generated diffusing molecule, whose accumulation in the surrounding environment determines a chemical gradient; the cells in its proximity respond enhancing the production of the mediator and activating a positive feedback, resulting in a coordinated response.
The first example of quorum sensing was discovered in Vibrio fischeri, in which the autoinducer is an N-acylhomoserine lactone (AHL), specifically N-(3-oxohexanoyl)homoserine lactone (3O-C6-HSL), and the response consists of the activation of bioluminescence; but there are many other activities that show a similar regulation, such as biofilm formation, virulence and sporulation. With our project, we aim to obtain different bacterial populations who, growing in the same environment, can activate a similar response: the different populations are engineered with different feedback boxes. When the mediator is present, translation is activated and the cross-talk makes the system auto-regulative.
References:
Brenner's Encyclopedia of Genetics (Second Edition), 2013, Pages 25–27, Quorum Sensing, S.P. Diggle, P. William
Molecular principles
In our work we’ve used two different kinds of Escherichia Coli’s strains, one encoding for a red fluorescent protein (RFP) and the other one for a green fluorescent protein (GFP). The molecular pathways for the expression of both fluorescent proteins are not independent but closely related, so that bacteria can “communicate” each other, a fundamental key of The Sound of Coli. In order to achieve these results, we’ve designed two plasmids (called SYS1 and 2 in the scheme). Every vector encodes just for one kind of fluorescent protein (GFP or RFP) and for different protein products that connect distinct E. coli colonies.
This vector contains fuor gene: luxR, tetR, GFP and las I; they are controlled by three promoters, pLac, pLux and pTet. In particular, luxR gene is located downstream of pLac, pLux controls both tetR and GFP and pTet is situated upstream of las I.
The molecular pattern’s goal
If AHL, a SIS 2’s product, is present in a bacterial colony transformed by a SIS2 plasmid, then GFP is produced and AI-1 synthesis is instead inhibited.
How it works in greater detail?
The constitutive promoter pLac promotes the expression of luxR gene. The protein LuxR coupled to AHL (produced by SIS 2) positively regulates the inducible promoter pLux, whose activation allows the transcription of tetR and GFP. TetR protein inhibits pTet’s activity, an usually active promoter that induces Las I expression. Las I is able to produce the metabolite AI-1, which is then secreted in the environment.
SYS 2
The plasmid's structure is analogous to SIS1's one, since it encodes for four proteins (LasR, LacI, RFP, LuxI) and it contains three promoters (pTet, pLas, pLac). The promoter TET controls the transcription of lasR, pLas is upstream both lac I and RFP, luxI upstream pLac.
The molecular pattern’s goal
Bacteria transformed with SIS2 normally produce AHL (acylated homoserine lactone): neverthless, the SYS2’s product AI-1 can inihibits AHL expression and promote RFP synthesis.
How it works in greater detail?
The constitutive promoter pTet actives the transciption of lasR, which in turn couples with AI-1 (a product of SYS 1) and promotes pLas, an inducible promoter which allows the expression of lac-1 and RFP. Lac I negatively regulates pLac, an promoter that is usually active and whose function is to promote luxI expression. Lux I protein is able to produce AHL.
Expected results
Using different quantities of the two strains, we should achieve a variety of concentrations of AHL and AI-1 and, in consequence, of GFP and RFP. In particular, what we expect is a “casual” oscillation of fluorescence intensity dipending on the number of bacteria in both types of Escherichia coli colonies.
Molecular effectors of quorum sensing
Quorum sensing (QS) is a cell-to-cell communication phenomenon that allows bacteria to control the expression of specialized target genes depending on their cell population size [1]. During QS process, bacteria produce a kind of signal called autoinductor (AI), which is secreted to the environment. When AI reaches the critical concentration, the consequence is the growth of bacterial population and the induction of changes in the gene expression, which in turn influences life cycle and bacterial metabolism. QS signal molecules (effectors of QS-dependent gene expression) are multifunctional molecules connected to life, development and death in single and mixed microbial populations [2]. The first described quorum-sensing system belongs to the bioluminescent marine bacterium Vibrio fischeri, and it is considered a model for quorum sensing in most gram-negative bacteria [3], which use the class of protein called AHL as signals [1]. V. fischeri colonizes the light organ of the Hawaiian squid Euprymna scolopes, where the bacteria grow to high cell density and induce the expression of genes required for bioluminescence. The light provided by the bacteria for counter illumination is fundamental to mask squid’s shadow and avoid predation; the bacteria benefit of this symbiosis too, because the light organ is rich in nutrients and allows proliferation more than seawater. Two proteins, LuxI and LuxR, control the expression of the luciferase operon (luxICDABE). LuxI is the autoinducer synthase, since it produces the autoinductor AHL (acyl-homoserine lactone); LuxR is the cytoplasmatic autoinducer receptor/DNA-binding transciptional activator. After its production, AHL freely diffuses in and out of the cell, increasing in concentration if cell density increases too. When AHL signal reaches a critical concentration, the molecule is bound to LuxR and this complex activates transcription of the operon encoding luciferase [3].
AHL are a class of molecules used by Gram-negative bacteria [1]; the Autoinducer I (AI-I) is also called N-(3-Oxohexanoyl)homoserine lactone and it’s a particular kind of AHL.
References:
1}Kumari, Anjali, et al. "Biosensing systems for the detection of bacterial quorum signaling molecules." Analytical chemistry 78.22 (2006): 7603-7609.
2}Gospodarek, E. U. G. E. N. I. A., T. O. M. A. S. Z. Bogiel, and P. A. T. R. Y. C. J. A. Zalas-Wiecek. "Communication between microorganisms as a basis for production of virulence factors." Pol J Microbiol 58.3 (2009): 191-198.
3}Waters, Christopher M., and Bonnie L. Bassler. "Quorum sensing: cell-to-cell communication in bacteria." Annu. Rev. Cell Dev. Biol. 21 (2005): 319-346.
Physical principles
Fluorescence
Fluorescence was described in scientific literature in 1845 by John Herschel, who observed a peculiar behavior by a colorless solution of quinine sulfate which, when exposed to sunlight, developed a blue surface color, but only in the 20th century came the knowledge of the sufficient atomic structure to describe the nature of the phenomenon and its first uses in spectroscopy represented by Aleksander Jabłoński's work.Fluorescence is the property of some substances to re - emit electromagnetic radiation received at greater wavelength and hence to lower energy, particularly to absorb radiations in the ultraviolet and emit it in visible. An incident radiation excites the atoms of the fluorescent substance, promoting an electron to an energy level (see orbital) less bonded, more energetic and therefore more "external". Within a few tens of nanoseconds, the excited electron returns to the previous level in two or more phases, for one or more excited states in intermediate energy. All decays except one are usually non-radiative, while the latter emits light at wavelength greater than the incident radiation (not necessarily in the visible spectrum): this light is called "fluorescence."Fluorescence is one of the radiative process with which relaxation of an excited molecule can occur. In fluorescence the luminescence ceases almost immediately after eliminating the exciting radiation. The radiation life time is short of the order of ns. In fluorescence radiation is generated by transitions between states with the same multiplicity of spin (eg S1 → S0). If a molecule with a wavelength radiation that matches exactly what is required to bring valence electrons from the fundamental level to the first excited level is lit, the radiation is absorbed and then resumed completely without any wavelength variation. If, on the other hand, the energy of the radiation is such as to energize the electrons to a level higher than the first, the system has the ability to return to the critical state in several stages, thereby emitting less energy than the incident light. In the molecules, the emission phenomenon does not occur without wavelength variation, since the excitation of electrons from one energy level to the other actually occurs as a transition from a band of rotational and vibrational levels associated with an electronic level to another level band associated with an excited level over the previous one. For this reason a polyatomic molecule absorbs a single frequency can emit a radiation band. The radiation bandwidth emitted is called Stokes or Antistokes radiation depending on whether it is of wavelength greater than or less than the absorbed radiation. Once the excitation from the fundamental electronic state to the excited one, with absorption of hν energy, decay occurs partially within the excited electronic level, following decay at a lower vibrational level.The energy of this decay is emitted in the form of thermal energy, while the electronic decay is accompanied by emission hν 'where, in this case ν' <ν.
Fluorescence in our experiment
In our experiment we used two different proteins with fluorescence properties Green Fluorescent Protein (GFP) is a protein expressed in the Aequorea victoria medusa. Thanks to its fluorescence property, its modest size and its ability to modify the spectroscopic characteristics within a certain limit, GFP has become a widespread tool for experiments and molecular biology techniques over the last decades. GFP, when struck and excited by radiation at a specific wavelength 488 nm laser line and is optimally detected at 510 nm., redirecting green light. Red fluorescent protein (RFP) is a protein derived from a coral. In biology it is considered a versatile marker for monitoring physiological processes, visualizing protein localization, and detecting transgenic expression in vivo. RFP can be excited by the 488 nm or 532 nm laser line and is optimally detected at 588 nm. The instrument we used to detect fluorescence values is called Tecan.
Team Unifi
unifi.igem@gmail.com
