Team:MichiganState/Notebook/Gene Engineering
Judging
Awards
Human Practices
Lab Notebook
May
Week 1
Tuesday
- Discussed Gene Engineering team goals
- Joined Benchling and walked through the basics of the site
- Planned to research different viable biocontainment mechanisms
Wednesday
- Reviewed information learned from literature search on biocontainment mechanisms
- Ruled out a thermal kill switch as an option due to fluctuating temperature of the bee abdomen
- Discussed what we learned about Snodgrassella alvi (optimal conditions, metabolic functions, etc.)
Thursday
- Discussed how the literature review revealed no metabolites unique to S. alvi that could be used for biocontainment
- Discussed possibility of using a T7 promoter and polymerase as a biocontainment mechanism and planned to research further
- Discovered that S. alvi has a Type 6 secretion system, which secretes products into other bacterial cells
Week 2
05/10 - 05/16
Monday
- After more research, we found S. alvi also has a Type 1 secretion system, which we could use for secreting our detoxification enzyme
- Discussed the Bee Microbiome Toolkit paper that successfully expressed plasmids in S. alvi
- Discussed the possibility that the T7 promoter idea may not work for biocontainment, as it was shown to have weak expression in S. alvi
Tuesday
- Researched codon optimization
- Did more research into Type 1 secretion systems
- Began working on a “Biocontainment Methods” document outlining the different ideas for biocontainment
Wednesday
- Discussed CRISPR genome integration, to be used for biocontainment
- Met with our graduate student mentor, Kati, and learned more about how CRISPR genome integration is performed in E. coli
- Discovered through our research that Type 1 secretion systems use a specific genetic tag to mark for secretion, as well as how we could use this in our design
Thursday
- Continued working on the “Biocontainment Methods” document
- Investigated the GhoST toxin-antitoxin system
- Located the S. alvi (strain wkB2) genome and inserted it into Benchling
Week 3
05/17 - 05/23
Monday
- Decided on the T7 promoter, combined with the GhoST toxin-antitoxin system as our method for biocontainment
- Identified open locations on the S. alvi genome that could be used for insertion of genes
- Researched different antibiotics with the goal of finding the one that would do the least harm in the environment near commercial farms
Tuesday
- Decided streptomycin antibiotic would be the best choice, as it is widely used in crop agriculture
- Continued research into the Type 1 secretion system and its applications in biotechnology
- Continued research into CRISPR use in E.coli
Wednesday
- Found the secretion genes present in S. alvi
- Met with Kati and learned more about how to use Benchling, as well as how to make primers on NEBuilder for Gibson Assembly
- Discussed measurement assays
Thursday
- Located plasmids from the Bee Microbiome Toolkit and added the ones we will use to Benchling
- Researched in more depth S. alvi growth conditions and metabolic pathways
- Met with graduate student mentor Shay and discussed using a Type 1 secretion system
Week 4
05/25 - 05/29
Tuesday
- Made a document outlining all of the plasmids we plan to assemble for our project, including all of the parts we need
- Located the hlyA secretion tag we will use and added it to Benchling
- Located and added all of the plasmids, promoters, and genes we will use to Benchling
Wednesday
- Discussed the different cloning methods, focusing on understanding Gibson Assembly in more depth
- Continued work on making list of all the plasmids to assemble
- Discussed the two different options for delivering gRNA into cells for genome integration
Thursday
- Determined we will use the Bee Microbiome Toolkit suicide vector that contains both the gRNA and gene to be inserted as our method for delivering the gRNA
- Discussed how gRNAs work and how to design them
- Discussed how to create and order gblocks
June
Week 5
05/31 - 06/06
Monday
- Fully assembled the chromosome integration plasmids in Benchling
- Fully assembled the secretion plasmids in Benchling
Tuesday
- After discussing with the whole team, decided that Gene Engineering will focus on designing a working secretion system in S. alvi
- Researched synthetic Type 1 secretion systems that have been used in bacteria besides E. coli
Wednesday
- Continued research into genetically engineered Type 1 secretion systems
- Researched different fluorescent proteins that could be used to test secretion
- Discussed how to order gblocks from IDT
Thursday
- Investigated different iGEM teams that have worked with the HlyA secretion system
- Continued research into fluorescent proteins
- Started talking about the experimental design of our project
Week 6
06/07 - 06/13
Tuesday
- Determined the secretion parts we will use from the iGEM registry
- Finalized the experiments we plan to conduct this summer
- Finalized the plasmids we will need to construct and order for those experiments
Wednesday
- Created a rough outline for our experiments
- Discussed with Kati how our constructs will be assembled
- Determined which parts will be synthesized and ordered, and which parts we will get from the lab
Thursday
- Learned how to assemble primers on Benchling
- Determined that restriction enzymes will be used for assembling our plasmid with multiple parts
Week 6
06/14 - 06/20
Tuesday
- Worked on the design of our constructs and primers
- Ordered S. alvi strain from ATCC
Wednesday
- Continued working on our constructs and primers in Benchling
- Discussed PAM sites with Kati and made subsequent changes to the gRNA in Benchling
Thursday
- Continued troubleshooting the design of our constructs
- Started working on the iGEM safety form
Week 8
06/21 - 06/27
Tuesday
- Completed the iGEM safety form
- Ordered vectors from Addgene
Wednesday
- Met with our faculty mentor Michaela to finalize all of our Benchling vectors, gblocks, and primers
- Made a few more changes based on feedback
Friday
- Doublechecked all of our constructs
- Ordered gblocks and primers from IDT
Week 9
06/28 - 07/04
Tuesday
- Researched the optimal conditions for culturing S. alvi
- Started gathering information about the different protocols we will need for our experiments
Wednesday
- Discussed how S. alvi requires 5% CO2 concentration to grow and how we will accommodate for this
- Discussed the different media S. alvi can grow in
Thursday
- Identified the protocols needed for our experimental design
- Assigned each team member different protocols to research and write up
July
Week 10
07/05 - 07/11
Tuesday
- Researched the different protocols
- Gathered more information about the different media S. alvi has been known to grow in
Wednesday
- Met with Kati to discuss protocols
- Discussed performing a growth experiment with S. alvi with different media
- Determined we would use a CO2 sachet for growing S. alvi in both liqud and solid media
Thursday
- Continued revising our protocols
- Researched all the different media for S. alvi
- Determined the recipes for different media and checked the availability of these reagents at Biochem Stores
Week 11
07/12 - 07/18
Tuesday
- Discussed the need for primers to amplify inserts in our plasmids to check that Gibson Assembly was successful
- Created these test primers on Benchling for our constructs
Wednesday
- Realized that a few of our previously ordered primers had some issues, so created new ones to replace them
- Ordered the replacement primers and the test primers from IDT
Week 12
07/19 - 07/25
Tuesday
- Discussed finding potential general detoxification enzymes to insert into our plasmid
- Started researching bacterial GSTs
Wednesday
- Finalized our experimental protocols with Kati and made some minor changes
- Decided we would test TSB and LB as media for S. alvi growth
- Discussed using R or Python software to analyze data
Thursday
- Discussed bacterial GSTs and determined they are not a good candidate for our project
- Walked through a tutorial of Python
- Decided to start research on insect GSTs that could be used as detoxification enzymes
Week 13
07/26 - 08/01
Tuesday
- Researched different insect GSTs
- Created a document outlining our findings about these enzymes
Wednesday
- Continued literature search of different insect GSTs
Thursday
- Discussed our insect GST findings
- Decided that we believe HAGST-8 from the insect Helicoverpa armigera to be the best candidate based on its experimental results with different pesticides and modeling done with imidacloprid in particular
August
Week 14
08/02 - 08/08
Monday-Friday
- Week off subteam work
Week 15
08/09 - 08/15
Wednesday
- Met with Kati and discussed progress on the experiments
- S. alvi had been resuscitated in TSB and grown overnight in the CO2 sachet
- PCR of the pBTK510 plasmid was unsuccessful and will be repeated
Friday
- Met with the Bioinformatics team to discuss researching oxidative stress responses in P. putida in order to determine if it produces one when exposed to imidacloprid
- Assigned team members different oxidative stressors to investigate
Week 16
08/16 - 08/22
Monday
- Researched oxidative stressors
- Met with the Bionformatics team to discuss our findings
- Ruled out certain stressors that could be used in an experiment, planned to do more research
Tuesday
- Continued research on oxidative stressors
Friday
- Participated in a Zoom experiment with Kati
- Observed and took notes while she performed PCR purification of the pBTK510 plasmid
- Observed and took notes while she performed a Gibson Assembly with the pBTK510 plasmid and the mRFP gblock
Week 16
08/23 - 08/29
Monday
- Observed and took notes while Kati transformed the E. coli strain WM6026 with the assembled plasmid
- Also watched Kati prepare media for the conjugation of the plasmid to S. alvi