Team:XMU-China/Applied Design

--------* Ideas *--------

Heavy metal pollution has long plagued the people. 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 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.

--------* Bio-Amplifier *--------

The detection limit we aim to achieve is quite low, so we designed a bio-amplifier which can vastly improve the expression of downstream gene, this means that we can use it to amplify signals produced by reporter gene, or enlarge the effect of enzymes by increase their amount.

T7 amplification systemIs the main feature of the amplifier system. The E.coli T7 system is regarded as the most widely used system for high-level gene expression. This system consists of a lambda DE3 lysogenic E.coli strain carrying a chromosomally integrated copy of the T7 RNA polymerase gene (gene 1) controlled by the lacUV5 promoter and a high-copy number vector allowing target gene expression from the T7 promoter. In contrast to other E.coli expression systems using host RNA polymerases for heterologous gene expression, an appropriate T7 system yields higher protein amounts since the bacteriophage RNA polymerase exhibits enhanced processivity.

T7 RNA polymerase is highly selective for its own promoters, which do not occur naturally in Escherichia coli. A relatively small amount of T7 RNA polymerase provided from a cloned copy of T7 gene 1 is sufficient to direct high-level transcription from a T7 promoter in a multicopy plasmid. Such transcription can proceed several times around the plasmid without terminating, and can be so active that transcription by E.coli RNA polymerase is greatly decreased.

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).

--------* Reporting device *--------

Since the basic idea of the hardware is to measure the fluorescent signal, Chemiluminescence signal or the electrical signal, we design the three different reporting system including the green fluorescent protein, lacZ and luxAB. The following paragraphs will discuss and compare the three important reporting genes.

a) LacZ gene is widely used in gene expression regulation in genetic engineering. LacZ gene encoding β-galactosidase (referred to as β-gal) is composed of four subunits of tetramer, can catalyze the hydrolysis of lactose. Beta-gal is relatively stable, with X-Gal as the substrate colored blue, easy to detect and observe.

This year, 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 follow 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℃. The mechanism of the production of electrochemical signal is:

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

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.

b) The green fluorescent protein (GFP) is a protein composed of 238 amino acid residues (26.9 kDa) that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm, which is in the lower green portion of the visible spectrum. The fluorescence quantum yield (QY) of GFP is 0.79. GFP makes for an excellent tool in many forms of biology due to its ability to form internal chromophore without requiring any accessory cofactors, gene products, or enzymes / substrates other than molecular oxygen.In cell and molecular biology, the GFP gene is frequently used as a reporter of expression.

c) Meanwhile, we also designed another reporter based on chemiluminiscence. Luminous bacteria are the most abundant and widely distributed of the light-emitting organisms and are found in marine, freshwater, and terrestrial environments. What their most important feature is they can produce the luciferase called LuxAB, which can catalyzes the bioluminescence reactions. Almost all luminous bacteria have been classified into the three genera Vibrio, Photobacterium, and Xenorhabdus.In our progress, the luciferase what 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 as follows:

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

The reduced flavin, FMNH2, bound to the enzyme, reacts with O2 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 reporter system, in heterologous hosts such as Escherichia coli.Eithe luxAB alone may be used (in which case decanal must be provided as substrate), or luxCDABE can be used, in which case the organism can synthesise aldehyde itself.Because the E.coli is capable for reduceing the FMN to FMNH2, so it is not necessary to add luxG as E.coli for the host.

--------* Use 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.