Heavy pollution has plagued the people for a long time. How to detect the concentration of trace heavy metal ions and how to decrease the detection limit as much as possible have become a challenging task. To solve this problem, a hardware-based system was designed in our project. The designed system can be applied into many areas to solve a variety of problems people are facing.

The pathway is as follows, the construction of a programmable cell can be divided into sensor, amplifier, reporter three modules. The components of each module have different function.



Based on this idea, our team designed a gene pathway model. When putting the pathway into Escherichia coli, the bacterium detector of trace ions formed.

I. The bio-amplifier

We designed a bio-amplifier which can vastly improve the expression of the downstream gene, this means that we can use it to amplify the signals produced by the reporter gene, or enlarge the effect of enzymes by increase their amount.

As we know, T7 promoter is one of the strongest promoter in prokaryote, but it cannot work if there’s no T7 RNA polymerase in the cell. Based on this theory, we designed our bio-amplifier, BBa_K2310000, as figure 1 shows, which is a special accessory that can be added into the gene circuit, between the RBS and the downstream gene.


Figure 1 The bio-amplifier

We tested our designed bio-amplifier with two different promoters, BBa_J33201, an arsenic induced promoter and BBa_K1334002, a formaldehyde induced promoter, and the results show that our amplifier is effective with a 1.3-1.6 times amplification factor.

Meanwhile, for this reason, we suggest that iGEM can develop another kind of BioBrick part called Amplifier, which is similar to existing kind of part, Inverter. With this new kind of part, synthetic biology can have another useful tool and it can also encourage more teams to develop other Amplifiers.

Accidently, we found that this amplifier can also be used for judging inducible promoters (to see more details about this application, you can visit our Measurement page).

II. New reporting device

We designed a hardware-based reporter based on electrochemical analysis, which is more accurate and faster than traditional reporters such as fluorescence proteins.

LacZ gene is widely used in gene expression regulation in genetic engineering. LacZ gene encodes β-galactosidase, which can catalyze the hydrolysis of lactose. Beta-gal is relatively stable, with X-gal as the substrate colored blue, easy to detect and observe, and so lacZ can be used with X-gal plates for blue-white selections.

But in our project, lacZ will not be used just for blue-white selections. Another substrate, PAPG, as figure 2 shows, can also be hydrolyzed by β-galactosidase, and one of the hydrolyzete, PAP, a small molecule which can be electrolyzed will be produced. This hydrolysis reaction is very efficient that only needs a 30-minute incubation at 37.


Figure 2 The mechanism of the production of electrochemical signal

To analyze the concentration of PAP, an electrochemical analysis will be taken by cyclic voltammetry on an electrochemical workstation with a three-electrode system.

Meanwhile, luxAB, a reporter which can produce chemiluminiscent signals, is designed as well.

Our results show that our electrochemical reporter works well and it is really efficient that it only need a total of 75 minutes to produce a cognizable signal.

Anyway, no need to worry if you do not have an electrochemical workstation. We have designed a hardware device called East-Wind, which contains a micro electrochemical workstation with electrodes for using this well-designed reporter. You can click here for more details about the East-Wind.

Meanwhile, we also designed another reporter based on chemiluminiscence. Luminous bacteria are one of the most abundant and widely distributed of the light-emitting organisms and are found in marine, freshwater, and terrestrial environments. Ttheir most important feature is they can produce the luciferase called LuxAB, which can catalyzethe bioluminescence reactions. Almost all luminous bacteria have been classified into the three genera Vibrio, Photobacterium and Xenorhabdus. In our project, the luciferase we use is from the Xenorhabdus luminescens.

LuxAB is a part of luxCDABEG which is the normal structure of the operon in most bioluminescent bacteria. The LuxCDE gene controls the synthesis/regenerate aldehyde and the FMNH2, which is provided by an FMN reductase such as LuxG. The LuxAB luciferase is a heterodimeric enzyme of almost 80kDa composed of α and β-subunits whose molecular weight is 42kDa and 39kDa. For the two subunits, the α subunit plays a major role which is responsible for the light-emitting reaction and the β-subunit is important for stabling the protein, although there is about 40% identity in the amino acid sequence between the α and β subunits.


The 3D-structure of LuxAB luciferase

The light-emitting reaction catalyzed by the LuxAB involves the oxidation of reduced riboflavin phosphate (FMNH2) and a long chain fatty aldehyde with the emission of blue-green light (490nm). This reaction is as follows:

FMNH2 + RCHO + O2 ===== FMN + H2O + RCOOH + hv (490 nm)

The reduced flavin, FMNH2, bound to the enzyme, reacts with 02 to form a 4a-peroxyflavin. This complex interacts with aldehyde to form a highly stable intermediate, which decays slowly, resulting in the emission of light along with the oxidation of the substrates.

There are two ways to use the LuxAB as a reporting system, in heterologous hosts such as Escherichia coli. Either luxAB alone can be used (in which case decanal must be provided as substrate), or luxCDABE can be used, (in which case the organism can synthesize aldehyde itself). Because E.coli is capable for reducing the FMN to FMNH, so it is not necessary to add luxG as E. coli for the host.

However, we’ve designed a LuxAB translational unit, BBa_K2310100, which can be used by only adding a promoter you want. We also tested this BioBrick carefully and particularly. To get more information, you can visit our Results page.

III. New methods to use your engineered bacteria conveniently

As we know, most of engineered bacteria can only work in the lab because they can only survive a short time with their specific engineered functions in most natural environment. Meanwhile, bacteria are usually stored in liquid medium or glycerin, which is too inconvenient for us to use them in the hardware. To ensure the practical applications of our engineered bacteria, especially in our hardware devices, a new method is needed.

Faced with this problem, we've designed a chip-based system to keep, store and use our engineered bacteria which is matched to our hardware device. In brief, our engineered bacteria can be freeze-dried through and stored in special-designed microfluidic chips.


The sandwich-like structure of the microfluidic chip

To learn more about this part of design, you can visit our Hardware page.