1. Sistemi za zaznavanje celične gostote v sintezni biologiji

2. Zaznavanje celične gostote (quorum sensing, QS)
Celice med seboj komunicirajo preko signalnih molekul, ki v drugi celici sprožijo odziv na ravni transkripcije.
Več ko je celic v bližini neke celice, ki jo opazujemo, več signalnih molekul jo bo doseglo. Informacija o gostoti celic je pomembna, ker celice v gosti kulturi (lahko) spremenijo fenotip.
Signalne molekule omogočajo sinhronizacijo fenotipa (npr. usklajen odgovor na imunski odziv gostitelja ali na spremembe v okolju, kjer rastejo). Med geni, ki se inducirajo, so tudi geni za sintezo signalnih molekul (sicer je njihovo izražanje nizko). Povečana koncentracija signalnih molekul torej sproži pozitivno povratno zanko.

4. Signalne molekule za zaznavanje celične gostote
Različni mikroorganizmi uporabljajo različne signale, ki so zanje specifični in jih torej signali drugih mikrobov ne motijo. Če zaznavajo več različnih signalov, se lahko na vsakega od njih odzovejo drugače. Obstaja tudi signaliziranje med celicami različnih organizmov (QS cross-talk).
Obstaja več skupin signalnih molekul, znotraj vsake skupine pa so razlike npr. v dolžini stranskih verig ipd. Signalne molekule nekateri na splošno imenujejo tudi avtoinduktorji, imajo pa jih tudi za bakterijske feromone.
for gram-positive bacteria

5. butirolaktoni (Streptomyces)
G-:
(acil)homoserinlaktoni (HSL) alkil-kinoloni (AQ) metilni sestri maščobnih kislin
G+:
peptidi
butirolaktoni (Streptomyces)

6. Odkritje QS
Princip QS so odkrili pri gojeni bakteriji Vibrio fischeri, ki sicer živi v svetilnem organu havajske pritlikave sipe Euprymna scolopes (pa tudi drugih mehkužcev in rib). Izražanje luciferaze je omejeno na kratko obdobje, ko celice dosežejo določeno gostoto (>1010/ml). Kasneje so podobne sisteme odkrili tudi pri številnih drugih bakterijah, obstajajo pa tudi načini komuniciranja med organizmi, ki so si filogenetsko zelo oddaljeni.

7. Signalni sistem LuxR/I
The receptor of the signal and the activator of the lux operon was shown to be a single protein molecule encoded by the luxR gene. The amount of luxR protein in a V. fischeri cell appears to be approximately 500 molecules. The luxR protein has two binding domains that interact with the autoinducer and the promoter region of the lux operon. The amino terminus contains the binding site for the autoinducer. This was elucidated by sequential amino terminus deletions of the protein, that eventually yielded an active transcription factor that was independent of the concentration of the autoinducer present. The carboxyl terminus was shown to possess a helix-turn-helix binding motif which typically interacts with DNA. Through careful experimentation it has been suggested that in the absence of autoinducer the amino terminus is able to mask the carboxyl terminus, preventing luxR from binding to the right lux promoter. LuxR has been localized to the cytoplasmic side of the plasma membrane in V. fischeri. However there does not appear to be a characteristic transmembrane a-helix in the luxR protein. It has been suggested that the amino terminus associates with the membrane while also masking the DNA binding site on the carboxyl terminus. The release mechanism of the luxR gene product for the membrane has not been elucidated, but proteolytic cleavage has been ruled out. However, it appears that once the autoinducer binds to the luxR protein, LuxR's carboxyl terminus is free to bind to the DNA because the amino terminus is released from the membrane and the DNA binding domain is no longer shielded. There are still many fine points to the luxR gene product that need to be elucidated, but its role in autoinduction is quite clear.
The autoinducer has been isolated and characterized for many species in addition to V. fischeri. The autoinducers are all chemically related compounds called homoserine lactones. However, there is a great deal of species specificity to autoinducers, meaning that one autoinducer is unable to convey a message to another species. The autoinducer in V. fischeri is referred to by two names in the literature, N-(3-oxohexanoyl) homoserine lactone and b-ketocaproyl homoserine lactone. The molecule is synthesized by the luxI gene product which is the first gene in the lux operon. Autoinducers are extremely mobile molecules that can easily escape the confines of the cell. They can then enter the surrounding media, and subsequently penetrate into another nearby cell. Therefore, in a limited volume, with a constant source of autoinducer production, the concentration of autoinducer rises. In a constant volume, the amount of autoinducer correlates to the amount of cells present that are producing autoinducer. Thus, it is clear how the autoinducer is a measure of the concentration of cells. Once the concentration of autoinducer reaches a critical threshold value, enough is present that the autoinducer will likely enter a neighboring cell. This concentration, where the autoinducer begins to successfully signal other cells, is between 5 and 10 nanomolar. Upon entering the cell, the autoinducer binds to luxR and forms a positive transcriptional complex. This positive transcriptional complex then activates the lux operon so as to increase transcription by approximately a thousand fold. Normally, luxI's gene product is at a basal level, and the cell thereby consistently produces autoinducer. However, when the lux operon is activated, even more luxI is produced, thus producing more autoinducer.

8. DocStock

9. Signalni sistem LuxS/AI-2 pri V. harveyi (a) in E. coli (b)
(a) Vibrio harveyi uses two sensor kinases, LuxN and LuxQ, to recognize AI-1 and AI-2, respectively. (b) In Salmonella and Escherichia colithe AI-2 receptor is the LsrB periplasmic protein.

11. Motilci QS
Ker je QS med drugim pomemben pojav pri patogenezi različnih bolezni, zato so razvili motilce tega procesa (proces imenujejo quorum quenching). Možni pristopi so z razgradnjo signalnih molekul ali z njihovo inaktivacijo.
Haptens used to prepare quorum quenching antibodies
Org. Biomol. Chem. , 2012, 10,

12. Inženirski pristopi k uporabi sistemov za zaznavanje gostote celic
S spreminjanjem ravni izražanja lahko dosežemo velike razlike v obnašanju sistemov. Najpogostejša načina regulacije sta preko spreminjanja promotorskih zaporedij in preko spreminjanja RBS. Slednje je pogostejše, ko gre za spreminjanje vezij, kjer bi lahko sprememba tipa promotorja porušila arhitekturo vezja. Spreminjanje promotorskih regij je smiselno pri tistih promotorjih, za katere imamo dovolj predhodnih eksperimentalnih podatkov o vplivu točno določenih mutacij. Spremenimo lahko tudi življenjsko dobo posameznih proteinov, če jim dodajamo oznake za razgradnjo.
PhD, 2010
General method for the construction and engineering of synthetic biological circuits. Multiple rounds of component selection and testing (optimization) are usually required to achieve desired circuit activities due to incomplete knowledge of component properties and interactions.

13. Za kaj lahko uporabimo sisteme za zaznavanje gostote celic?
Z uporabo naravnih in prilagojenih sistemov lahko dosežemo usklajeno izražanje točno določenih genov v populaciji tarčnih celic, ko te dosežejo ustrezno gostoto – smiselno npr. pri izražanju toksinov, za izvedbo logičnih vrat, za klinične aplikacije.
Prednost uporabe teh sistemov je tudi v tem, da lahko zakasnjeno izražanje sprožimo v oddaljenih celicah (oddajniki  sprejemniki).
Inputs and outputs of a basic LuxI/LuxR quorum-sensing module. The response of a LuxI/LuxR quorum-sensing module can be controlled by regulating the expression levels
of either the LuxI or LuxR proteins. Output from the quorum-sensing module is in the form of transcription from PluxI.
Establishment of intercellular signaling between distinct cellular populations. Signal generation in sender cells is dependent on the output of a hybrid phage λ lambdatetO
promoter (PLtet0-1) regulated by the Tet repressor. Upon generation, OHHL diffuses through media to receiver cells, activating LuxR and causing expression from PluxI.

14. We designed an Intelligent quorum regulating system contains three devices to make cell density decrease moderately and control the apoptosis happen in a certain part of cell. The first device is for quorum sensing. The second device is for moderation and targeting and the third device leads to programmed cell death.
When the density of AHL is high enough, AHL will combine with a protein called LuxR and become a complex. This complex can inhibit downstream gene, TetR. So the next device will not be inhibited anymore and then begin to work.

15. Vezje 2: oscilator Vezje 3: apoptozna naprava
We added an oscillating circuit as the moderate device. In this circuit, three proteins inhibit the expression of each other. The function of oscillator is outputting the CI periodically which leads the accumulation of mazeF. In other words, the accumulation of mazeF is moderated.
The CI expressed in oscillation device will activate PCD device. Once CI is expressed, the expression of MazE will be inhibited. No more hexamer will be formed, and MazF will show its power and the density of cell will decrease. When the density is too low to activate the system, then there will be no CI. After that, the MazE will be expressed again.

16. Generic Metric to Quantify Quorum Sensing Activation Dynamics
ACS Synth. Biol., Article ASAP
DOI: /sb400069w
Publication Date (Web): September 6, 2013
Quantifying QS activation in synthetic gene circuits. (A) The QS genes are carried by plasmid pLuxRI where LuxR and LuxI are under
IPTG control (in LacI+ strains). pluxGFPuv acts as the reporter plasmid for QS wherein the GFP variant GFPuv is produced under the control of
the PLuxI promoter. (B) QS circuit with feedback on signal synthase luxI. (C) MG1655 cells grown in TBK media with 1 mM IPTG at 30 °C were
observed periodically using flow cytometry for GFP expression. Cells carried either pLuxRI and pluxGFPuv (green:QS) or pLuxR and pluxGFPuv
(gray) as luxI negative control. The histogram of GFP fluorescence at different time points after dilution starting from the overnight culture is shown.
QS controlled fluorescence initially decreases to the same level as control cells but starts to increase again at high density (OD > 0.1). Shaded
rectangles on the left mark regions where fluorescence decreases (gray), stays low or off (pink), and increases (yellow). (D) Continuous highthroughput
monitoring of culture density (OD) and culture fluorescence (normalized to OD) in plate reader alongside the flow measurement in C
shows the QS controlled transition from OFF (pink region) to ON (yellow region). Blue arrow marks the corresponding point at which GFP
expression was first visible by flow measurement in C. The initial decrease seen in flow measurement in C (gray) is not visible here, since the initial
dilution density is below OD based detection. The transition from OFF to ON (pink to yellow) takes place in the same OD range as observed with
flow cytometry. Colored circles depict the mean values while error bars are the standard deviation measured from three technical replicates.