Team:Stanford/Notebook

Lab: Biosafety & Notebook

Our lab situation was a complicated one from the start. Due to the pandemic, we were not (and have never been) permitted onto Stanford's campus to use the Teaching Labs as we would have normally. While we could have scaled back our project to something either solely or heavily computational instead of choosing a project that requires substantial wet lab work to run, we were very motivated and excited for this project in particular, and decided we would do whatever it took to get into a lab space of some kind. Our quest to find lab space took us all over - from looking into outsourcing work to graduate students, to using a friend's garage lab, to automating the work through cloud labs such as ECL. Eventually, we ended up finding lab space in a biohackerspace / community lab in Santa Clara called Biocurious, where four of our team members were able to work for some length of time.

Biosafety at Biocurious

Prior to beginning work in BioCurious, all team members that worked at BioCurious had completed prior training for lab work on Stanford campus. All of our team members that worked in BioCurious had had existing certified training in General Safety, Biosafety, Life Science Safety, Chemical Safety, Compressed Gas Safety, and Bloodborne Pathogens Safety. Additionally, in order to begin work in BioCurious, the specific project our team was working on had to be approved by administrators at the lab. All work in BioCurious must be BSL-2 or lower to be approved to work in the space. Prior to beginning work, all members were required to have watched all safety training videos for BioCurious, as well as score a 100% on a required safety quiz prior to entering the building. For COVID-19 specific precautions, everyone working in BioCurious is required to wear a face mask at all times, remain 6 feet apart from other members, wipe down surfaces after use, and not reuse any disposables, including gloves. Members working at BioCurious are required to sign in, sign a safety acknowledgement, and no more than 15 people are allowed in the building at once. Non-members are not permitted entry to prevent the spread of COVID-19. While in lab, all of our researchers practiced all necessary safety precautions for dealing with bacteria, chemicals, nucleic acids, and compounds. 

The organism we are working with (B. subtilis) is a generally-regarded-as-safe (GRAS), BSL-1, gram positive bacteria. Naturally found everywhere from the soil to the human gut microbiome, it is a non-pathogenic, extremely safe bacteria. When working with B. subtilis, our team still took all precautions, handling only with gloves, and adding bleach to cultures before disposing of them in a biohazardous waste container.  Any materials that may have come into contact with the bacteria were cleaned with bleach before disposal into a hazardous waste container. All genetic constructs ordered and used by the team contained no viral or pathogenic sequences. All chemicals used were disposed of properly in accordance with their safety sheet. Researchers wore gloves, long pants, and close-toed shoes at all times in lab. When necessary, researchers used lab coats, safety goggles, the fume hood, and alternate types of gloves.

Considering Biosafety in Our Project

While developing our diagnostic, safety has been one of the most important considerations throughout its design, and was part of the reason that chose to use Bacillus subtilis. An ideal use case for our product involves at-home testing that can be performed by anyone, or even eventual integration into the human body as a biological threat monitor. When designing SEED, the health and safety of the user was at the forefront of our minds. We chose Bacillus subtilis due to its status as a GRAS, BSL-1 bacteria. It is already found naturally in the human microbiome, and is non-pathogenic. In an at home setting, accidental exposure to this bacteria should have very little risk to the user. For our homologous recombination detection method, it takes advantage of a natural system found in B. subtilis, meaning the risk of harmful side effects to the bacteria are low. Furthermore, by using a negative selection marker as the readout, cells that do not uptake the target are killed, and do not pose a threat to the user. Cells that do encounter the target and survive could have additional built in genetic mechanisms that will kill them and their lineage after a certain amount of time or when desired to prevent dangerous mutations. For the safety of our researchers, when performing all our experiments, B. subtilis was always kept on plates with antibiotics to prevent infection.

Lab Notebook: Weekly Summaries

We have organized our lab notebook here by week instead of day. This is mostly because our lab work was sporadic and lagged behind at times, due to the way we had to receive our supplies. Instead of getting reagents and cells directly shipped to Biocurious for us to receive, everything had to be sent to our mentor's lab at Stanford and we had to coordinate with grad students to organize pickup times to come get what we had ordered. This bureaucratic process ultimately slowed the pace of our work.

Week 1: September 13-19

• Revived B. subtilis strains received from the Bacillus Stock Center (1A976 and 1A1276): Protocol #1
• Mini prepped pBS1C: Protocol #2
• Tested competence of 1A976: Protocol #4
• Took inventory and figured out what reagents we still needed to order
• Verified antibiotic resistances: We plated untransformed B. subtilis on kanamycin, chloramphenicol, and erythromycin plates as a negative control which. Plates were made by adding a 1000x dilution of the antibiotic stocks to standard LB plates. 

Week 2: September 20-26

• Attempted gibson assembly onto faulty pBS1C backbone: Protocol #5
• Transformed gibson results into E. Coli (got nothing): Protocol #6
• Tested competence of 1A976: Protocol #4

Week 3: September 27-October 3

• Continued trying to miniprep (pBS1C): Protocol #2 
• Ran gels to confirm presence of pBS1C: Protocol #7
• Transformed miniprepped pBS1C into 1A976: Protocol #4
• Ran experiment to see if E. Coli were growing: we inoculated a 5mL tube containing nutrient broth with a colony from our E. Coli plate. We then allowed them to grow overnight and checked the OD in the morning

Week 4: October 4-10

• Ordered new minipreps from Qiagen and Zymogen
• Ran experiment to test for sensitivity of DNA uptake by 1A976: we used Protocol #4, but adding differing amount of DNA to the cells (1 microgram, 500ng, 250ng, and 50ng of DNA)
• Got new cells containing pBS1C from the Bacillus Genetic Stock Center and mCherry and YFP plasmids from Dr. Elizabeth Libby of the Silver lab

Week 5: October 11-17

• Mini Prepped everything (mCherry plasmid, YFP plasmid, pBS1C): Protocol #2
• Gibson Assembly of ManP and Toehold Plasmids: Protocol #5
• Ran gel to test for assembly: Protocol #7
• Transformed manP and Toehold Plasmids into competent  E. Coli: Protocol #6

Week 6: October 18-24

• Miniprepped E. Coli clones containing the assembled ManP and Toehold Plasmids in preparation for sequencing: Protocol #2. Tested ManP and Toehold constructsSent for sequencing via Elim BiopharmaceuticalsSelective plating on the antibiotic that they should have received resistance for via the assembled plasmids
• Miniprepped many colonies of pBS1C, mCherry plasmid, and YFP plasmid in preparation for large competence experiment: Protocol #2
• Tested competence by varying several parameters including time allowed for competence induction (1hr, 2hrs, and 3hrs), the temperature the cells grew at (37 degrees Celsius, and ~23 degrees celsius/room temperature), and agitation (cells were either shaken or not shaken): all combinations of these parameters were transformed via a protocol based on Protocol #4, but with requisite changes based on the variable conditions they were put under (i.e. if there were kept at room temperature, they were not put back into the shaker, but rather left on the bench at room temperature)
• Tested ManP and Toehold constructs;
  - Sent them for sequencing via Elim Biopharmaceuticals
  - Selective plating on the antibiotic that they should have received resistance for via the assembled plasmids

Week 7: October 25-Wiki Freeze

• Ran sporulation procedure: Protocol #8. Verified competence experiment from week 6 using the same procedure
• Verified competence experiment from week 6 using the same procedure