In the Laboratory
At the beginning of summer everyone on our team took the online UC Fundamentals of Laboratory Safety course. This course described correct use of personal protective equipment and engineering safety controls within the lab to reduce risks associated with each lab space. The course also covered methods involved in chemical safety including the identification of chemical hazards through the use of Material Safety and Data Sheets (MSDS) and pictograms as well as proper chemical storage and disposal. In addition to this general safety course, we were also trained on the laboratory specific hazards by the lab managers of the three spaces that we worked in.
Since our project involved use of some proteins found in known pathogens, we decided to synthesize the genes we were interested in through IDT to express in laboratory E. coli. These safety precautions ensured that we 1. did not culture pathogenic organisms in the laboratory and 2. the proteins we expressed were not toxic or pathogenic. To ensure that our engineered strains did not exit the lab, we killed all of our cultures with 10% (v/v) bleach before disposal. We also did not transport any engineered strains outside of the lab, reducing the risk of release.
Our project involved the use of an array of small molecules. To ensure safety, we followed recommendations in the MSDS for each of the chemicals that we used. When ordering our library of alternate FabI substrates, we made sure not to order chemicals that were acutely toxic. For the chemicals we tested, we followed the MSDS recommendations for proper use of personal protective equipment including, wearing eye protection, lab coats, and wearing rubbing gloves, and engineering controls such as a working in the fume hood to eliminate the risk of chemical exposure.
The inhibitor we are detecting, triclosan, is toxic, so we created a triclosan waste container to avoid dumping triclosan down the drain. The proper way to dispose of this container is to give it to UC Davis’s Environmental Health and Safety Services
In the Real World
The use of laboratory expressed enzymes in the environment is defined in The Coordinated Framework for Regulation of Biotechnology . Our device and its associated biosensor must be approved through this process before use in the real world. Additionally, for application in the real world setting, future screening of alternate substrates would require us to look for substrates that are non hazardous to use outside of the laboratory setting. The handling of known concentrations of triclosan must be handled with proper PPE outside of the lab. See above for triclosan disposal.
Preparation of DNA for Cloning
Originally, our cloning method was to be Gibson assembly. We ordered our 8 genes with 5’ and 3’ gibson overhangs from IDT. Once received, we rehydrated the genes:
1. Centrifuge for 3-5 seconds at >3000 x g to pellet at the bottom
2. Add 20 uL TE buffer or Milli-Q water for a final concentration of 10ng/uL
3. Briefly vortex and centrifuge
4. Store at -20 degrees for up to 24 months with TE buffer or 1 month with dH2O
We made four 50 mL and four 2 mL overnight cultures of DH10B containing pET29b+ vector for insertion with kanamycin resistance selection. The grown 50 mL cultures were then transferred to 50 mL falcon tubes and spun down to pellet at 4000 x g for 10 minutes. We then followed the protocol included in the Invitrogen MaxiPrep kit used. The results gave an average of 1.93 ng/uL, which is below the threshold for accurate concentration measurement for the Biotek Epoch spectrophotometer with the Take3 Microvolume plate. This also showed that the results yielded next to no DNA for the MaxiPrep and decided to go forth with a MiniPrep on the four 2 mL cultures using the Invitrogen MiniPrep protocol provided with the kit. The results yielded an average of 90 ng/uL which is above the 60-80 ng/uL concentration needed for the restriction digestion.
Gel separation and purification:
Checking for colonies and sequencing:
Transformation for Expression:
Checking for colonies and glycerol stock for culture growth:
Cell growth for expression:
Expression and Protein Purification: