SAFETY

SAFE LAB WORK

Safety is an essential part of any lab project in science. Proper safety regulations and precautions lower the risk of accidents. They are designed to protect the experimenter, the project, the public and the environment. At ETH-DBSSE it is mandatory for every person to take a Safety Course. The course is held by Mr. Niels Buerckert, who is the Technical Manager and head of the department’s Safety Committee. During the Safety Course we first covered basic safety measurements required for both office and lab members of the department. We were instructed how to handle hazardous situations in case of natural disaster or accident. We got acquainted with locations of fire extinguishers, fire blankets and emergency exits. In the case of a small fire, the fire blanket is used. In the case of a bigger fire, the CO2 fire extinguisher is used. If paper or carton is on fire, foam extinguisher is used instead. We were instructed who to call in the case of fire and how to act and who to call in the case of an injury. After we considered different specific situations in basic safety training, we covered lab safety measurements and regulations.

Lab safety measurements tackle every aspect of lab work. The experimenter has to wear proper clothing which covers as much skin as possible. Shorts and open shoes are not allowed. Additionally the experimenter needs to wear a lab coat, gloves and wear goggles on the top of the head. Food and drinks are not allowed in the lab. Smoking is forbidden. We were instructed how to properly label hazardous chemicals and how to act in the case we encounter unfamiliar or unlabeled chemical. In the case where we encounter unknown chemical in solid form we use a brush to gently remove the chemical and dispose it in hazardous waste. Any unlabeled liquid is considered as a potential hazardous chemical. We got acquainted with the specifics of waste management in this building: the location of clean room, how to properly transport chemicals from one part of the building to another, how to prevent potential spread of chemicals from our lab to other areas of the building. Chemicals need to be transported in the chemical bucket. When transferring chemicals from one floor to another, they are not allowed in the personel elevator. The experimenter must send the chemical separately in the elevator for chemicals, while the experimenter uses the personel elevator. Gloves need to be removed after exiting the lab. If the chemicals require handling in gloves, the experimenter must use one hand without a glove to interact with the surroundings (opening doors etc) and one hand with a glove to carry the chemicals. We were taught how to properly maintain a clean lab space and how to correctly store chemicals in the lab to prevent accidents. Every sink has an eye shower and every floor has emergency showers in case a person gets in contact with a hazardous chemical.

SYSTEM OVERVIEW

Figure 2: System Overview

figure 2

DESIGN MOTIVATION

One major challenge in synthetic biology is the difficulty to combine modular frameworks into higher order networks that are reliably reproducible.We designed our circuit to be as modular as possible. As the interest of this genetic circuit mainly lays when multiplexing is possible, we designed it such that you can associate any candidate signal just by changing the promoter. Thus, constructing a simple library of AHL, you can create a library of E. Coli capable to sense a wide range of microbiota signals. Moreover, our model shows that the system is easily tunable playing with NorR, Esar, and integrase production and degradation rates to fit to the required range of detection, and thus would be the best solution for a modular multiplexing investigation tool.

INPUTS SENSOR

We need a NO sensor and a AHL sensor. Later we will also need a Lactate sensor, as recent researches tend to asses that lactate is present in extremely high quantity in some heavy case of IBD in children. Our input sensor is composed of a PnorV promoter associated with esaboxes situated downstream the promoter and upstream the reporter gene. In an improved version of this sensor, the esabox (to which EsaR can bind and act as a roadblock , preventing gene transcription) will be put in different place around the PnorV promoter. The idea is to see if competitive binding can bring better result than traditional independent gene activation and inhibition. More over it is known that PnorV activation mechanism under NO/NorR binding involves DNA looping aroung the promoter. As a consequence, a low efficiency of EsaR road block behavior is expected as the looping could prevent it from binding to the esaboxes.

SWITCH

We currently developed three different possible switches, based on integrase kinetic, CRISP/Cas9 and the recently discovered CRISP/Cpf1 complex. When both NO and AHL are present the hybrid promoter is activated and lead to intregrase protein production. Intregrase is a protein that is capable of binding to a particular DNA sequence referred as AttP and AttD. After binding the DNA sequence is cut and inverted. The switch acts here as a memory bit that can be flipped from 0 to 1 in an irreversible way. The flipped sequence contains a constitutive promoter associated with esaboxes, and thus negatively regulated by EsaR.

REPORTER

On each side of the flipped sequence are placed a mNectarine and a GFP gene respectively. Depending of the flipping state of the DNA sequence, the cell produces either mNectarine of GFP. As explained above this gene expression is regulated by ,EsaR. Thus once the DNA is switched, the cell produces GFP if AHL is present in the medium.

GENETIC CIRCUIT

Figure 3: Genetic Circuit

figure 3

NO SENSOR

We use here the PnorV promoter, specific to NO sensing. We also use a constitutively produced NorR protein. NorR exist under a dimer form in the cell. Each dimer is able to bind to the 3 binding site present on PnorV. Then those three dimer assemble in a hexameric ring like structure. When NO is present in the medium, it binds to the structure and activate the promoter.

AHL SENSOR

In addition to the PnorV promoter, we had downstream esaboxes. Esar is constituvely produced in our bacteria. Just like NorR it exists as a dimer in the cell. The dimer form binds to the esaboxes forming a roadblock and unabling gene transcription when NO only is present. When AHL enter the cell it binds to the EsaR dimer and free the promoter, allowing transcription.

LACTATE SENSOR

Recent studies highlighted the fact that Lactate seems to be over-present in some very heavy case of IBD, especially in children. Thus it is also interesting to investigate the role of Lactate in IBD occurrences. We uses here a modified version of the Plac promoter. two LldR binding sites O1 and O2 are situated upstream the promoter. LldR and LldD (Lactate -> Pyruvate catalyst) are constitutively produced. in absence of Lactate, LldR binds to O1 and O2 forming a DNA loop and preventing transcription. When Lactate enter the system, it binds to the LldR dimer and free the promoter. We introduced LldD to increase the threshold of Lactate sensing.

AND GATE

The AND gate is the association of both NO sensor and AHL or Lactate sensor. It is constituted by a hybrid promoter composed of the PnorV promoter and the downstream esaboxes. Model shows that playing with NorR and EsaR production and degradation rates can allow a fine tuning of the sensor to adapt to the wished detection range.

SWITCH MODULE

AND gate activation triggers integrase production. Its role is to inverse the DNA strand containing the GFP gene.

REPORTER MODULE

the reporter module is just constituted of some esaboxes and the GFP gene. Under AHL presence, the reporter (GFP) is expressed.

CONCLUSION

One major challenge in synthetic biology is the difficulty to combine modular frameworks into higher order networks that are reliably reproducible. We designed our circuit as interchangeable, easily tuneable parts. This allows multiplexing, one of our circuit’s major advantages for a non-invasive, modular, multiplexing, in vivo tool to investigate IBD’s unknown causes.

Team:ETH Zurich/Safety