Team:NCTU Formosa/Safety

A centrifuge is a kind of instrument generally driven by an electric motor. By rotating in very high speed, it produces a big centrifugal force perpendicular to the axis and rotates an object around a fixed axis. A centrifuge is used to separate the components which have different molecular weight in a mixture. The centrifuge works with the sedimentation principle, where the centripetal acceleration causes denser substances to separate out along the radial direction (the bottom of the tube). Conversely lighter objects will tend to move to the top⁽¹⁾. In our lab, we usually separate E.coli from LB broth and then conduct mini-prep with it.

The orbital shaker incubator is widely used in microbiology labs, cell biology labs, etc. It provides a stationary temperature to cultivate bacteria and cells. Some incubators also have function to maintain the humility level. In our lab, the incubator provides stationary temperature at 37 °C, which is the optical temperature for E.coli to grow, and 16 °C for ligase enzyme working⁽²⁾. Our team was authorized to work with an non-pathogenic bacterial strain, the Escherichia coli DH5. The strain is regularly employed in research, industry and study. Biosafety Level 1 standard regulations were strictly followed while using this strain.

An autoclave is a pressure chamber used to sterilize equipment and supplies by subjecting them to high pressure saturated steam at 121 °C for around 15–20 minutes depending on the size of the load and the contents. The autoclave is used for the safety issue. When sterilizing, the pressure and temperature inside reached high level, forming superheat steam and high pressure which will kill cell and make proteins destroyed or denatured⁽⁴⁾.

In our lab, every consumables that may contact bacteria will be sterilized before and after use to guarantee the safety.

A laminar flow is an enclosed, ventilated laboratory workspace for safely working with materials contaminated with (or potentially contaminated with) pathogens requiring a defined biosafety level. We usually conduct experiments in which E.coli will expose, such as picking single colony⁽⁷⁾. We strictly rule that all of the researchers should conduct experiments carefully inside the laminar flow and sterilize his/her hands with alcohol solution (70%) before experiments, all of us are familiar with the using method of Laminar flow.

An analytical balance is a class of balance used in measuring small mass in the sub-milligram range. The measuring pan of an analytical balance is inside a transparent enclosure with doors so that dust can be collected well and so any air currents in the room do not affect the balance's accuracy⁽⁵⁾. In our lab, we have two models of balance, one is accurate to 0.1 g, another is accurate to 0.1 mg.

In this system, water is purified in a first step using unique Jetpore ion-exchange resin, synthetic activated carbon and UV lamp emitting at 185 and 254 nm to reach a resistivity of 18.2 MΩ.cm at 25 °C and a TOC value below 5 ppb, both monitored by advanced analytical techniques⁽⁶⁾. The water purify system is used to produce ultrapure water, which is for experimental use in our lab.

The thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus that are most commonly used to amplify segments of DNA via the polymerase chain reaction (PCR). The function of thermal cycle is to provide the appropriate working temperature for some reactions, including many temperature-sensitive reactions such as restriction enzyme digestion or rapid diagnostics. The device has a thermal block with holes where tubes holding the reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps, and time setting determines the time interval of every temperature⁽³⁾.

Gel documentation system, also known as a gel image system or gel imager, is equipment widely used in molecular biology laboratories for the imaging and documentation of nucleic acid and protein suspended within polyacrylamide or agarose gels. These gels are typically stained with ethidium bromide or other fluorophores such as SYBR Green. Generally, a gel doc includes an ultraviolet (UV) light transilluminator, a hood or a darkroom to shield external light sources and protect the user from UV exposure, and a CCD camera for image capturing⁽⁸⁾.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (called SDS-PAGE) is a technique for separating proteins based on ability to move within an electrical current, which is a function of the length of their polypeptide chains or of their molecular weight. By adding SDS detergent to remove secondary and tertiary protein structures, the proteins maintain as polypeptide chains. The SDS coats the proteins, mostly proportional to their molecular weight, and confers the same negative electrical charge across all proteins in the sample. To help focus the proteins into sharp bands at the beginning of the electrophoretic run, the Laemmli system is widely used, which uses tris-glycine gels comprised of a stacking gel component and the resolving gel where varying acrylamide gel percentages are used to separate the proteins based on their mass weight⁽⁹⁾.

We conducted the polyacrylamide gel electrophoresis to check whether E.coli can express our PBAN device correctly.