Team:SCUT-China A/Project Design
DESIGN
As the pollution of heavy metal is serious, and the current methods of detection
are complex and expensive, this year SCUT-China_A team want to develop an efficient and low-cost method
to detect heavy metal ions.
Our idea is inspired by the release of neurotransmitters from synapses. When
a nerve impulse is transmitted to the postsynaptic membrane, the calcium ion channels would be opened,
so that the synaptic vesicles would be transported out of postsynaptic membrane and release the neurotransmitters.
Because the neurotransmitter is pre-synthesized, when the impulse arrives, the membrane is able to respond
immediately and quickly. In similar way, we make an effort to combine the lysis gene and a kind of "chemical
signal factor" to form a report module which can quickly respond to specific "impulse".
Aimed at imitating the release of neurotransmitter, we looked up some researches about the mechanisms of the phage’s infection on E. coli: when the phages infect E. coli, the bacteria’s cell wall would lysis, so that the viruses could be released. We got the lysis gene SRRz from the phage, and tried to standardize it to build up a bacteria lysis module. The results indicated that This lysis module could cause rapid lysis of E. coli, and the expression of lysis gene is much more efficient than previous reporters such as GFP.
We decide to look for a kind of "chemical signal factor" that can serve as our system’s
“signal factor” (like the “neurotransmitters” in nerve cells). For the purpose of having obvious and
clear phenomenon, an endogenous enzyme which can lead to chromogenic reaction would be our ideal choice,
because the catalytic reaction of the enzyme is rapid and even a small amount of enzyme could result
in clear phenomenon. What's more, we can he calculate the amount of enzyme relatively accurately by measuring
the consumption of the substrate or the production of the product.
We found that
endogenous enzyme β-galactosidase from the E. coli, which is able to act as the "chemical signal
factor" in our chromogenic module. It can catalyze colorless substrate such as x-gal or oNPG and produce
blue or yellow products. The results indicated that with the IPTG induction, the gray value of the result
of chromogenic reaction present an obvious law which can be used in quantitative measurement.
As the pollution of heavy metal is serious, and the current detection methods are complex and expensive, we try using our repot module to detect heavy metal ions. In order to specifically detect heavy metal ions, we refer to mechanisms and genes related to bacteria's metal tolerance, from which we find promotors and regulatory factors can specifically respond to heavy metal ions. We selected five operons that are able to respectively respond to Hg 2+ , Pb 2+ , Cd 2+, Ni 2+, Cr 6+. And we finally develop three sensitive and specific metal ion detecting module (mercury detecting device(BBa_K2360008), cadmium detecting device(BBa_K2360010) and lead detecting device(BBa_K2360011)).
Based on the results of the previous researches, we are going to synthesize five kinds of operons which can
specifically respond to certain heavy metal ions and place them on the upstream of the SRRz lysis gene.
At first, the bacteria would stably express the β-galactosidase. Once the bacteria accept a "signal"
—— specific heavy metal ions, it would express the lysis gene rapidly so that the bacteria would be lysed.
Therefore, the β-galactosidase could be released into the solution with substrate and lead to chromogenic
reaction.
The depth of the color（which is reflected as its gray value） produced by the chromogenic reaction is
positively correlated to the lysis rate of the cells, and the lysis rate of the cells is positively correlated
to the intensity of the SRRz, which is induced by its operon, and finally determined by the intensity
of the heavy metal ions. This result made it able to be used for not only qualitative detection but also
quantitative measurement.
If possible, based on the principles mentioned above, we will develop a mobile phone app to optimize
our method of quantitative measurement. People can use our devices to qualitatively detect whether there
are heavy metal ions in the solution, and use their cell phone to scan the sample after the chromogenic
reaction to quantitatively measure the intensity of the ions.
