Abstract

The mechanism of Sensor A engineering bacteria

This gene circuit applies bistable mechanism, involving two types of promoters: recA and lac, which dividedly regulate the expression of the gene sequence of repressor lacl and lexA, whose product suppress promoter lac and recA. The reporter gene of lacl and lexA thereafter bring out RFP(red fluorescent protein) and GFP(green fluorescent protein), both of which could ~~~~. As upstream and downstream gene promote and mutually repress, the whole system has two stable output state:

State A: As promoter precA works, repressor lacl is expressed, then the product lexArepresses promoter lac, and repressor lexA is not expressed, thus cannot exert depressant action to promoter precA. When promoter recA works while promoter plac is shut down, the whole system remains perfectly stable. Afterwards, the downstream reporter gene of promoter precA, GFP, is turned on, then product can be detected.

State B: promoter plac works, repressor lexA is expressed, then the product lexA represses promoter precA, and repressor lexA is not expressed, thus cannot exert depressant action to promoter plac. When promoter plac works while promoter precA is shut down, the whole system remains perfectly stable. Afterwards, the downstream reporter gene of promoter plac, RFP, is turned on, then product can be detected.

Two external input signals: IPTG and radiation-produced SSB can separately counteract the inhibition of expression product of repressor lacl towards promoter plac and repressor lexA towards precA. Their involvement could make the whole system switch between the two states.

SensorA

The mechanism of Sensor A engineering bacteria

This gene circuit applies bistable mechanism, involving two types of promoters: recA and lac, which dividedly regulate the expression of the gene sequence of repressor lacl and lexA, whose product suppress promoter lac and recA. The reporter gene of lacl and lexA thereafter bring out RFP(red fluorescent protein) and GFP(green fluorescent protein), both of which could be output signals that represent the current state of the system. As upstream and downstream gene promote and mutually repress, the whole system has two stable output state:

State A: As promoter precA works, repressor lacl is expressed, then the product lexArepresses promoter lac, and repressor lexA is not expressed, thus cannot exert depressant action to promoter precA. When promoter recA works while promoter plac is shut down, the whole system remains perfectly stable. Afterwards, the downstream reporter gene of promoter precA, GFP, is turned on, then product can be detected.

State B: promoter plac works, repressor lexA is expressed, then the product lexA represses promoter precA, and repressor lexA is not expressed, thus cannot exert depressant action to promoter plac. When promoter plac works while promoter precA is shut down, the whole system remains perfectly stable. Afterwards, the downstream reporter gene of promoter plac, RFP, is turned on, then product can be detected.

Two external input signals: IPTG and radiation-produced SSB can separately counteract the inhibition of expression product of repressor lacl towards promoter plac and repressor lexA towards precA. Their involvement could make the whole system switch between the two states.

SensorB

Sensor B is a low-dosage-radiation-sensitive biological part, which involves both radiation response and quorum sensing. Sensor B applies a mechanism, completely from that of Sensor A.

The whole circuit is made up with SoxS P-LuxI-LuxPL-LuxR-LuxPR-LuxI-LuxPR-LasI, and LasI works as the final report gene produce protein PAI1 which is the signal of amplifier part. Sensor B can be divided into three functional units: Sensing, Amplifying, Reporting,4321bp long in all.

The first unit consist of SoxS P-LuxI, while SoxS P is a oxidation stress-sensitive promoter. When cell gets radiated, it will produce an oxidation stress, which induces SoxS P and further promote luxl expression. As a result, AHL synthetase is produced, it will then catalyze and synthesize autoinducer information molecule AHL.

The second unit composes of LuxPL-LuxR-LuxPR-LuxI. Generally, LuxPL gives a weak constitutive expression of luxR. When cell got radiated, protein LuxR integrates with autoinduer AHL and forms a complex, which subsequently increases the transcriptional rate of the lux pR promoter while also decreasing the transcriptional rate of the lux pL promoter. While the repression is much slighter generally. LuxPR starts the expression of Luxl, and then comes to produce more autoinducer AHL. In combination with LuxR, AHL thus increases the production of AHL. The whole unit is a positive feedback loop.

The third unit contains LuxPR-LasI, working as a reporter unit in this sensor. Through the interaction and amplification in the first two parts, the complex will exert effect on promoter LuxPR and induce the expression of LasI.

Amplifier

In sensor, the amount of promoter expression stimulated by external condition can be detected by the expression amount of fluorescent protein, but most of all invisible. So, in order to increase it several times of growth, we need amplification effect. In common practice, quorum sensing, which is a grouping bacteria behavior regulation mechanism, is always applicable. It shows that bacteria respond to colony density and environmental change by sensing autoinducer. When the number of bacterial colonies reaches a certain quorum, it starts a series of gene expression to regulate group reaction. At the very beginning, we originally applied lux system in Vibrio Fischeri, for its mechanism is relatively simpler but innovatively harder. Then, we switched to las system and rhl system in pseudomonas aeruginosa.

In the plasmid of amplification system, two activating transcription factors are in constant expression and remain in a certain concentration. With a certain level of autoinducer PAO1, pao1 integrates with lasR, the complex of which are bond to the target site of las promoter, then activating it and control the expression of the downstream reporter gene GFP in accord with the concentration of the amount of combination. In addition, this complex can also work on the promoter rhl as transcriptional activator of rhl system, and afterwards activating it.
The efficiency of complexes activating two promoters are relatively different. Theoretically, it is more efficient for the complex to take care of its own promoter than to work on the thl promoter as a transcription activator.
Actually, las and rhl system are not orthogonal, while las system disturbs the rhl system. PAO1 can not only bind to the lasR protein, also thlR protein, even in a very low level. Furthermore, the complex PAO1-rhlR activates promoter to express RFP.

The efficiency of complexes activating two promoters are relatively different. Theoretically, it is more efficient for the complex to take care of its own promoter than to work on the thl promoter as a transcription activator.

Actually, las and rhl system are not orthogonal, while las system disturbs the rhl system. PAO1 can not only bind to the lasR protein, also thlR protein, even in a very low level. Furthermore, the complex PAO1-rhlR activates promoter to express RFP.

Micro-fluidic chip

Material

PDMS glue; glass; semipermeable membrane (specifics are as follows)

Sensor bacteria produces AHL molecules when receiving radiation, and transferred AHL to amplifier bacteria, which gives out fluorescent light, whose intensity can be detected by measuring the light intensity.

Based on the simplified mechanism, the basic function of micro-fluidic chip contains:

1/separate culture of sensor and amplified bacteria

2/deliver the metabolite from sensor to amplifier, and mix it with amplifier bacteria as much as possible

3/fluorescent light given out by bacteria B can be detected.

4/detect multiple samples at a time

Design of chips

Single channel pattern

And the chip model to meet the needs of multiple data at one time is as follows.

Through making model, pouring, punching and bonding, a perfect chip is made up.

Function

Sensor bacteria above receives radiation, and metabolizes AHL molecules, which pass the semipermeable membrane and transmit the amplifier to the detection hole.

The transmission method remains unknown for we cannot make sure of the transmembrane way of AHL.