Team:HZAU-China/Description

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index

Inspiration

Objective of our project

How to use our machine

• Smell Sensing Module
• Memory Module
• Reproduction Module
• Erasure Module

Future work

. Inspiration

Facing the restriction of technologies, we've been accustomed to those things so-called "impossible" but have forgotten that science is originated from imagination which contains unlimited possibility. Maybe, someday, we can ask children for their childlike innocence and listen to their wishes. Being inspired, many astonishing and creative projects will thrive.

Just like our project, the inspiration of it came from a little wish of a child. We still remember that he attended a public engagement activity named "Little Future Scientists" held by our team in June this year. We prepared a series of educational and meaningful experiments for the activity, hoping that every child could enjoy himself/herself while learning something. After all experiments were completed, he watched the video that recorded the experiments that day on his father's mobile phone while reviewing the names of the instruments and the colors and the properties of the reagents. However, when he mentioned the smells of such things, he hesitated and said, "How magical these smells are! If I had recorded those smells, it would have been better."

. Objective of our project

To achieve this child's dream, we designed our project this year. We aimed to build a biological machine that can sense, memorize and reproduce smells. To make the machine reusable, we also design a memory erasure module that can recover the system to its initial status. The core functions can be realized by a precisely designed engineered E. coli strain. And we can build a hardware as a container and controller of the bacterium. In order to make the machine more human-friendly, we also design software on mobile phone to control the hardware.

In order to illustrate that the features of recording and replaying smells can work well, we take benzyl alcohol as an example. We design four modules for our machine, smell-sensing module, memory module, reproduction module and erasure module. We will use benzyl alcohol to verify whether each module will work.

Now, we're going to introduce each module briefly.

. How to use our machine?

 o  Smell-sensing Module

To start with our machine, simply press the button "Start Recording" on the interface of our software. Then, the machine will absorb the air around, and passing it to bacterial suspension. The smell molecules from the air will initiate the smell-sensing module.

The smell-sensing module consists of a gene for encoding a smell-sensing regulatory protein and an operon with a corresponding promoter that can be controlled by regulatory protein. After combined with the smell molecule, the regulatory protein possesses activity and initiates the operon expression by binding with the promoter region. The activation of the promoter will produce another signal which will initiate the memory module.

  Note 1.1: Smell-sensing operons

Bacterial operons play important roles in the regulation of bacterial physiology. With the regulation of these operons, bacteria can live more efficiently.

On account of these facts, we can utilize those operons in bacteria that responds to substances with smells in the environment as sensors.

 o  Memory Module

How do we know whether the bacterial cells have memorized the smell it just sensed? Simply pressing the button "View Status" on the interface of our software will tell! The photoreceptor of the hardware will inspect on the bacterium. If the receptor detects that the indicator which we placed in the bacterium has emitted the proper signal, the software will notice you with "Recording Complete". If not, it will notice you with "Recording in Progress".

The memory module is based on lux quorum-sensing system originally from Vibrio fischeri [1]. The most remarkable feature of the quorum-sensing circuit is its positive feedback. In our project, the positive feedback will cause the accumulation of a small RNA, taRNA [2]. We regard the accumulation of taRNA as the memory of the bacterial cells.

  Note 2.1: Bacterial quorum-sensing

Quorum-sensing is such a strategy that a bacterium can take advantages of the power of its populations in complicated environment. It is population-dependent regulation. The downstream genes controlled by quorum-sensing will not be expressed if the density of the bacterial cells is low. However, when the density of the bacterial cells is high enough, the downstream genes will be expressed [3,4].

The quorum-sensing circuit we used in our project is induced by an auto-inducer molecule named AHL (N-Acyl-Homoserine Lactone) [1]. It was found that there is a positive correlation between the population of cells and the AHL concentration in microenvironment.

The most typical feature in the bacterial quorum-sensing is its positive feedback circuit. The positive feedback circuit will continuously accumulate products of interest and will maintain the product on a certain level. We regard the accumulation of the product as what the bacteria has memorized, and this is the basis of our project.

 o  Reproduction Module

You can control the status of the reproduction module by pressing the button "Reproduce". After you press this button, a signal will be added into the media by the hardware, and switch on the reproduction module.

The signal along with taRNA forms a logic AND gate. The activation of this AND gate will trigger the initiation of the reproduction module [2]. The synthetase of the smell molecule will be translated and synthesize the target products.

  Note 3.1: Production of smells

Secondary metabolism in plants and bacteria can produce substances that produce smells, whether pleasant or unpleasant. A great many researchers working on metabolic engineering have found out how these substances are synthesized and successfully transformed these pathways into E. coli or optimized those pathways already existing. For example, the MVA pathway is usually used as the pathway producing isoprenoids and the optimized shikimate pathway is usually used as the pathway producing aromatic compounds. In our project, we can utilize those fine-optimized pathways to reproduce the smells that the bacteria have recorded [5,6].

  Note 3.2: Riboregulator

Riboregulator is a compound noun consisting of Ribosome and Regulator. Riboregulator is a pair of small RNA, taRNA and crRNA, which can regulate translations of proteins of interest. Riboregulator along with inducible promoters can form an AND gate which provide a memory reproduction switch for our project. The bacteria will accumulate taRNA as the memory molecules in our project. And when we want to reproduce its memory, we just need to add another signal to induce the promoter that transcribe genes of interest and switch on the memory reproduction circuit [2].

 o  Erasure Module

How to recover the machine to its initial status? Simply press the "Erase" button! By pressing this button, the hardware will add a signal into the media to initiate the erasure module.

The erasure module contains an AiiO gene for encoding acyl-homoserine lactone acylase. When the AiiO enzyme is induced, it will hydrolyze AHLs, which will quench the positive feedback of the quorum-sensing circuit. In this way, the memory of the machine is erased [7].

  Note 4.1: Quorum-sensing quenching

To switch off our memory circuit which is based on the quorum-sensing circuit, we need to quench the positive feedback of the quorum-sensing circuit. Naturally, the quorum-sensing was quenched along with the decrease of the bacteria population [3]. But in our project, we utilize an enzyme named acyl-homoserine lactone acylase, AiiO, to hydrolyze the AHL which is the auto-inducer of the quorum-sensing circuit. After AHL is hydrolyzed, the positive feedback will be quenched, and the system will recover to its initial status.

. Future work

At present, we take benzyl alcohol as an example to test our design. In the future, we're going to build a set of E. coli that can sense and reproduce different molecules with different smells, respectively. With this design, the assay can sense and reproduce various and complicated smell. The factual basis of this design is that many researchers have reported numerous operons that can sense molecules with smells. Researchers working on metabolism engineering have also managed to synthesize these smells in E. coli.

Reference

[1] Egland K A, Greenberg E P. Quorum sensing in Vibrio fischeri: elements of the luxI promoter[J]. Molecular Microbiology, 1999, 31(4): 1197-1204.

[2] Isaacs F J, Dwyer D J, Ding C, et al. Engineered riboregulators enable post-transcriptional control of gene expression[J]. Nature Biotechnology, 2004, 22(7): 841.

[3] Pai A, Tanouchi Y, You L. Optimality and robustness in quorum sensing (QS)-mediated regulation of a costly public good enzyme[J]. Proceedings of the National Academy of Sciences, USA, 2012, 109(48): 19810-19815.

[4] Mukherjee S, Bassler B L. Bacterial quorum sensing in complex and dynamically changing environments[J]. Nature Reviews Microbiology, 2019: 1.

[5] Pugh S, McKenna R, Halloum I, et al. Engineering Escherichia coli for renewable benzyl alcohol production[J]. Metabolic Engineering Communications, 2015, 2: 39-45.

[6] Liao P, Hemmerlin A, Bach T J, et al. The potential of the mevalonate pathway for enhanced isoprenoid production[J]. Biotechnology Advances, 2016, 34(5): 697-713.

[7] Li Y, Zhao J, Quan C, et al. Production, characterization, and powder preparation of quorum quenching acylase AiiO for pathogen control[J]. Preparative Biochemistry and Biotechnology, 2019, 49(2): 151-157.