Biosafety

General safety in our lab

Firstly, in order to be allowed to work in the lab, everybody had to take part in a lab tour, when our lab manager introduced us into the safety and work rules. The following main parts were included in it:

• Work with hazardous compounds

Hazardous substances are those that have injurious effect on health and environment. For example such substances can cause eye and skin irritation and damages or induce cancer. Hence in order to prevent ourself, our lab mates and the nature special handling and storage place is necessary. Taking care of the appropriate clothes and protection during the work with these compounds is also important.

• Personal Safety Instructions

Lab coat, gloves and glasses were compulsory to wear during every experiment, what we have done. However special glasses are requirements when UV light and blue light are used. Such glasses are provided in our lab, near the certain instruments.

• Accident risks during the work

The main accident risks can be mechanical injuries, burns and corrosion, poisoning, and electric shocks.
• Cutting: Never use damaged glasswear! Glass should be collected in designated boxes to avoid injury to the cleaning personnel.
• Burns and corrosion: Bunsen burners have almost invisible and silent flame so it is a hazard. The most common accidents in a laboratory is fire causing flammable solvents. Since our department work with large amounts of such liquids it is important that all person is aware of how to handle such a fire.
• Poisoning: It can occur by inhalation of gases or fumes, or through the skin.
• Electric shocks: If a person has gotten in contact with an AC line there is a severe muscle convulsion that makes the person unable to get loose from the line. Such cases are extremely dangerous.

First cell cycle - the daughter cell resetter

Our system was designed in order to “reset” the age counter in newly born daughter cells. To accomplish this, one of the key components of the age counter, dCas9, is not produced in the very first cell cycle of daughter cells. As a consequence, the gRNA molecules that leak into the daughter cell during cell division won’t be able to induce the production of any fluorescent protein. As can be seen in fig. 1 we expect only Csy4 to be produced in the first cell cycle. To be noted, the G1-specific degradation tag inserted in both Csy4 and dCas9 is expected to trigger the respective degradation of the species at the end of the G1 phase. This feature can also be seen in fig1, in which Csy4 is rapidly depleted after the end of G1 phase.

Our daughter resetter not only impedes the production of fluorescent proteins induced by gRNA-dependent promoter but it also produces YFP, which becomes the characteristic color of the first cell cycle, together with the signal needed for the expression of CFP during the next cell cycle. As showed in fig. 2, during the first cycle YFP is constitutively produced by the endogenous daughter-cell specific promoter PDSE4. By design, together with the mRNA for YFP, also the 28-gRNA1-28 is produced. This is the signal the will last until the second cell cycle and, after being processed by CSY4 and bound to dCas9, will be able to induce the production of CFP. One non-ideality included in the model is the fact that CSY4 is present during the first cell cycle (fig. 1); as a consequence some 28-gRNA1-28 are turned into gRNA1 at the very moment of their creation. We had no way to test experimentally how long either form of gRNA lasts in the intracellular environment; this is the main source of uncertainty in our model. For the sake of simplicity, we chose a parametrization that would allow for both gRNA molelcules to survive degradation until the next cell cycle.