Team:BIOSINT Mexico/Sensor

Modular Sensor

Description

There has been an increasing necessity to implement new, efficient and inexpensive techniques for the identification of biological and chemical agents that contaminate the environment, one of the most developed strategies for solving this trouble is the use of biosensors. A reporter device must be an easily detectable mechanism for sensing a specific substance of interest, this reporter needs to have a monitoring and a resettable capacity.

In this project the target pollutant that will induce gene expression of the biological sensor is the mercury, this is based on the loss of green pigmentation in Arabidopsis thaliana. A substantially faster loss of chlorophyll is needed if is used as a reporter system for a plant sentinel. Chlorophyll loss in plants is normally a slow process that occurs during the complex mechanism of senescence. The half-life of chlorophyll has been estimated to be 2–5 days for relatively mature and fully greened leaves (Stobart and Hendry, 1984), visual perception of chlorophyll loss in leaves can take longer.

The process for whitening the plant requires following the method explained below takes around 48 hours in order to see a complete change in color. In order to accomplish this it is necessary to use specific enzymes which degrades the chlorophyll that is already in the plant and also a doubled stranded RNA which inhibits the production of an important molecule involved in the production of chlorophyll (Medford, J. et al , 2006) De-greening).

Biosynthesis

The first phase of the chlorophyll biosynthesis starts with the glutamic acid, after nine chemical steps this amino acid produces a four ring structure, called protoporphyrin IX. A molecule of magnesium is add to the ring structure by the magnesium chelatase, through two more steps this is converted in monovinyl protochlorophyllide and is reduce to chlorophyllide a. by the enzyme protochlorophyllide oxidoreductase (POR). The chlorophyllide a. is transformed in darker green chlorophyll and lacks the hydrocarbon tail.

The chlorophyllide molecule is converted to the darker green chlorophyll molecule by the enzyme chlorophyll synthetase, which adds a twenty-carbon phytol tail. Like most biological molecules, steady state levels of chlorophyll are maintained by a combination of biosynthesis and catabolism with the half-life of chlorophyll in a green plant being approximately 50 hours

Carotenoids act as accessory pigments in photosynthesis and form the basic structural units of photosynthetic antennae. They also serve as photo-protection agents by quenching singlet oxygen that might otherwise damage chlorophyll. (Qu,L. et al, 2007)

Phytoene desaturase (PDS; EC 1.14.99.-) represents one of the key enzymes in the carotenoid biosynthetic pathway and is present in nearly all types of plastids in plants(Li, L. et al, 2008).

An inducible gene silencing system using intron-containing, inverted repeat RNA. (interference RNA). The silencing gene is PDS, because of the phytoene desaturase enzymes blocks carotenoids synthesis culminating in a photobleaching phenotype because of photo-oxidation of chlorophylls. For example, a time of 6–9 days was necessary for a photobleaching phenotype to become evident in Arabidopsis plants (Guo et al., 2003).

De-greening

If a characteristic white phenotype is going to be used to identify methyl mercury presence, it must be much faster than previous RNAi circuits. Medford, J et al (2006) proposed the combination of the following regulatory circuits for de-greening:

a) Stop synthesis circuit; induction of diRNA to reduce protochlorophyllide oxidoreductase (POR) or diRNA to lessen GENOMES UNCOUPLED 4 (GUN4).

POR is light-dependent enzyme that catalyzes the conversion of protochlorophyllide a to chlorophyllide a. GUN4 is a single copy gene that regulates chlorophyll biosynthesis by activating magnesium chelatase, a key enzyme complex that produces magnesium protoporphyrin IX, basic structure for chlorophyll. With a double-stranded interfering RNA construct designed for each one of them and placed under the control of certain promoter, A. thaliana demonstrates chlorophyll loss. (Antunes et. al 2006).

(b) Initiate breakdown circuit; induction of chlorophyllase (CHLASE) and red chlorophyll catabolite reductase (RCCR) or CHLASE and pheophorbide a oxygenase (PAO).

Chlorophyll breakdown involves a series of enzymatic steps. Key processes are the hydrophobic tail removal by CHLASE, red chlorophyll catabolite reductase (RCCR) and porphyrin ring cleavage by PAO.

By combining both processes de-greening is easily recognized within 24-48 hrs of induction.