Team:UCSC/Notebook

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Notebook

We precisely tracked all Progenie activities including preliminary research, human practice interviews, wet lab experiments, modeling progress, and collaboration meetings... down to the date and who participated. Provided is a transparent timeline of our work that leaves no room for confusion in attributions and allows for a full picture of our growth and learning.

  • Week of Mar 1 - 6

    Met with Dr. Amir Zarrinpar (UC San Diego) to discuss microbiome engineering potential (Stephen, Tobin)

  • Week of Mar 7 - 13

    Attended Chardan’s virtual 3rd Annual Microbiome Medicines Summit to ask industry experts and senior scientists about our plan for microbiome engineering (Rhea)

  • Week of Mar 28 - Apr 3

    Team completed 1) Hazardous Waste Management and 2) Laboratory Safety for Research Personnel as safety training courses before getting into lab (Whole Team)

  • Week of Apr 4 - 10

    • First day Tuesday lab team met up in-person to start project brainstorming/protocols (Rhea, Tara, Emily, Denise, Tanya, Yi-Chi, Wen Franklin)

    • First day Thursday lab team met up in-person to start project brainstorming/protocols (Tobin, Nabil, Rose, Fonz, Stephen, Torrey, David, Julia)

  • Week of Apr 18-24

    Practiced running and loading gels (whole team)

  • Week of Apr 25 - May 1

    Practiced making of chemicompetent C41 cells (whole team)

  • Week of May 2 - 8

    • Practiced making of electrocompetent BL21 cells (whole team)

    • CAHS1 transformation to test C41 chemicompetent cells.

    • Meeting with Dr. Jennifer Brophy (Stanford University) to discuss the use of the ICE system. (Tobin, Stephen, Torrey, Rose)

    • Meeting with Dr. Peter Turnbaugh (UCSF) to discuss microbiome engineering potential (Tobin, Stephen, David)

    • Meeting with Dr. Raman Khanna (UCSF) to discuss potential/plan for targeting and lowering LDL cholesterol (Rhea)

  • Week of May 16 - 22

    • First Biolink Depot meet

    • Meeting with Dr. Rodems (Santa Cruz DPC) to discuss antimicrobial resistance and current detection methods (Fonz)

  • Week of May 23 - 29

    Assigned groups to people for researching different project elements:

    • Conjugation: Denise, Tara, Rhea, Torrey

    • Lambda Red: Franklin, Tanya, Nabil, Fonz

    • Enzymes (PfAgo, Cas9, Cas12): Rose, David, Stephen, Tobin

    • Gene Drive: Emily, Julia, Wen, Yi Chi

  • Week of May 30 - Jun 5

    • Research presentations for various project ideas we brainstormed (Whole Team)

    • Meeting with Dr. Harris Wang (Columbia University) to discuss use of the MAGIC system. (Stephen, Tobin, James)

  • Week of Jun 20 - 26

    • Literature review into identifying the source of STEC, identified that beef and leafy greens are the most impacted, most STEC outbreaks occur in the Americas (Fonz)

    • Learned about the economic impact of STEC outbreaks, found out that distributors take the biggest financial toll (Fonz)

    • Literature research revealed that STEC transmission can increase up to 25% during the transportation of cattle to processing plants (Fonz)

    • Emailed Dr. Arie Haavelar (University of Florida Emerging Pathogens Institute) about the global impact of STEC. (Rose)

    • Worked on and finalized preliminary iGEM Safety form (Torrey, Tanya, Rhea, Nabil, Emily, Fonz)

  • Week of Jun 27 - Jul 3

    • Tested chemicompetent C41 cells with CAHS1 plasmid

    • Meeting with Dr. Joji Muramoto (UCSC) to discuss soilborne pathogens (Fonz, Emily)

    • Meeting with Professor Gregory Gilbert (UCSC) to discuss E. coli contamination and transmission pathways (Rhea, Emily, Franklin)

    • Meeting with Dr. Alison Weiss from the University of Cincinnati to discuss Shiga Toxin structure and function, and recombinant protein development. (Tara)

    • Worked on and submitted iGEM Grant for $2500 (Rhea, Torrey)

    • Prepared interview material for meeting with Taylor Farms’ food safety contact, Drew Mcdonald (Fonz, Julia)

    • Meeting with Taylor Farms for understanding how the produce industry works against food pathogens (Fonz, Denise, Rose, Julia)

    • Filmed promotional video (Rose, Julia, Franklin, Emily, David, Denise)

  • Week of Jul 4 - 10

    • Meeting with Robert Rust (Vaxxinova) about STEC vaccines (Yi-Chi, Torrey)

    • Shot reporter one scene and beginning plate scene for promo video. (Rose, Wen, David)

    • Meeting with Pomponio Ranch to understand how local ranches work to prevent STEC (Wen)

    • Took individual photos for team (everyone)

    • Meeting with SmartWash Solutions to understand what types of testing and prevention is done to stop STEC (Rhea, Nabil, Rose, Julia, Denise)

    • Filmed reporter two and reporter three scenes for promo video (Franklin, Rose, Stephen, Yi-Chi, Tanya, Rhea, Nabil)

    • Meeting with Birko Corporation to understand what types of testing and prevention is done to stop STEC (Rhea, Denise, Wen)

    • First time making KAN and CAM plates (Tobin, Rose, Torrey)

  • Week of Jul 11 - 17

    • Meeting with industry professional to understand how the meat industry tests and prevents STEC (Tara)

    • Reached out to Sternberg lab (Columbia University) to explore use of the INTEGRATE system in our project. Corresponded with graduate student Leo Vo (Tobin, David, Stephen)

    • Made captions and translations for captions for promo video. (Yi-Chi, Rose, Rhea)

    • Journal club: “CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome Engineering” by Vo et al.

    • Meeting with Pacific International Marketing to understand what their company does to prevent STEC outbreaks (Fonz)

    • Meeting with Dr. Josh Kittleson (former member of Anderson Lab at UCB) to discuss viability of P1 phagemids (Tobin, Stephen)

  • Week of Jul 18 - 24

    • Started researching other gram-negative bacteria after learning from the interviews that our project could have a broader application (Fonz, Denise, Rose, Julia, Emily, Rhea)

    • First meeting with IISER-K: Team Member Introductions, Project Introductions w/presentations, Gold Medal Requirements (what we each want from the other team and what the best way to collaborate is), Next Steps (making a discord, making a collaborative google folder, scheduling next meeting) (Whole Team)

    • Initial meeting with UC Davis iGEM team

    • Reviewed email from anonymous food safety contact that detailed preventative measures taken during the meat processing chain (Fonz)

    • Plated C41 and DH5alpha cells in order to make chemicomp cells. (Rose, Julia)

    • Acquired plate of E. coli WM3064 from Dr. Chad Saltikov (UCSC) for conjugation experiments (Torrey)

    • Meeting with Stanford Team to discuss potential collaboration (Rhea, Torrey, Tara, Stephen, Emily)

  • Week of Jul 25 - 31

    • Identified cronobacter sakazakii, campylobacter (Cdtb gene), vibrio cholerae, and yersinia enterocolitica as possible targets for Progenie (Fonz, Denise, Rose, Julia, Emily, Rhea)

    • Meeting with Mike Taylor (ex-USDA and FDA) to understand how the government tackles STEC outbreaks and prevention methods (Fonz, Julia, Rose, Denise, Yi-Chi)

    • Reached out to Sternberg lab (Columbia University) for pSL1812 (pSPAIN) aliquot.

    • Attempted to make starter cultures of DH5alpha, C41, and WM3064 for Inoue chemicomp protocol. (Nabil, Rose, Emily, Rhea, Fonz)

    • Meeting with Dr. Rodems over the phone to discuss the application of Progenie to other pathogens (Fonz)

    • Second meeting with IISER-K: they helped us with our population modeling aspect (gave suggestions to improve and wrote out some equations). We proposed a wet lab collaboration aspect, and the IISER K team sent over a list of action items that they would like to get done in our wet lab. Next Steps: figuring out which wet lab work we could take on for them, assigning a few members of our team to help them with our modeling (Rhea, Torrey, Stephen)

    • Grew up bacterial stabs of pE-FLP, the various pOSIPs, pSPIN, and the plate of WM3064 (Torrey, Tara)

    • Attempted to grow overnight cultures of DH5alpha and C41. Restarted WM3064 starter culture for Inoue chemicomp protocol. (Nabil, Rose)

    • Met with IISER Kolkata to discuss modelling

    • Grew up liquid cultures of the new plates of pE-FLP, the various pOSIPs, pSPIN, and WM3064 (Fonz)

    • Harvested chemicomp C41 and DH5alpha cells. (Nabil, Rhea)

    • Meeting with Dr. Matias Fingermann (Administración Nacional de Laboratorios e Institutos de Salud (Argentina)) about STEC vaccine (Yi-Chi, Tanya)

    • Created Inoue chemicomp SOP. (Nabil, Rose)

    • Miniprepped pOSIP-KL-mCherry, pE-FLP, and pSPIN (Nabil, Rhea, Yi-Chi, Fonz)

    • Made glycerol stock of DH5a transformed with the various pOSIP plasmids, DH5a transformed with pE-FLP, and the WM3064 cells

    • Performed initial pOSIP-KL-mCherry transformation and tested competency (Nabil, Rhea, Emily)

  • Week of Aug 1 - 7

    • Third meeting with IISER-K: further discussion about modeling and refining the math, deciding which wet lab work could be done by us, and how much IISER K would need to do on their own. Next Steps: Make an introduction to collaboration post on Instagram (Rhea, Torrey, Stephen)

    • Officially started wet lab collab with IISER-K (Tanya, Julia)

    • Meeting with Chad Saltikov to discuss conjugation experiment design (Torrey, Tara)

    • Meeting with Manel Camps to discuss plasmid incompatibility and mobilization - confirmed that we can simply add an F oriT to mobilize our plasmid with the native F plasmid! (Tara, Torrey)

    • Design chalk talk on mCherry clonetegration (Whole team)

    • Miniprep of CAHS1 transformed into WM3064, DH5a, C41 to test competence (Nabil, Fonz)

    • Transformed pSPAIN into NEB DH5a, CAHS1 into WM3064, C41, and our DH5a (Nabil)

    • Made chemicompetent WM3064- and C41-mcherry integrants (David, Tanya)

    • Finalized design for INTEGRATE target guides (Stephen, David)

    • Design chalk talk on using INTEGRATE

    • Miniprep of pE-FLP (Rhea, Yi-Chi)

    • Transformed pSPAIN into NEB DH5a (Rhea, Tobin)

    • First attempt to transform pE-FLP into C41-mCherry cells (Yi-Chi, David)

    • Met with IISER-K team to discuss the concepts behind their project and how we should help (Tanya, Julia)

  • Week of Aug 8 - 14

    • Made chemicomp WM3064-mCherry (Tanya, Julia)

    • Made pE-FLP overnight culture for miniprep (Rhea)

    • Made pSPAIN index plates and overnight culture for miniprep (Rhea)

    • Second attempt to transform pE-FLP into C41 pOSIP-mCherry (David, Yi-Chi)

    • Miniprepped the pSPAIN from 12 isolated & grown colonies (Tara, Rose)

    • Ran gel electrophoresis on the minipreps of 12 pSPAINs, pE-FLP, and CAHS1 (Torrey, Yi-Chi)

    • Design chalk talk on phagemid design, phage production, and infection experiments (whole team)

    • Meeting with IISER-K team to discuss wet lab collaboration work (Tanya, Julia)

    • Fourth meeting with IISER-K: modeling meeting (Intro to Simbio / Python Code, running through equations and making changes) (Rhea, Torrey, Stephen)

    • Third attempt to transform pE-FLP into C41-mCherry cells - pE-FLP found to be incompatible with native plasmid of C41 (pLys) (David, Yi-Chi, Tara)

    • Transformation of pOSIP-KL-mCherry in DH5a (David, Franklin, Yi-Chi, Fonz)

    • Transformation of pE-FLP into DH5a (David, Franklin, Yi-Chi, Fonz)

    • Made an initial plan for the wet lab collaboration work (Tanya, Julia)

  • Week of Aug 15 - 21

    • Attempted pSPAIN site directed mutagenesis to add guides to create five pMINT plasmids (Tobin, Stephen)

    • Attempted plate reader analysis of WM3064-mCherry and C41-mCherry (Stephen, Tobin)

    • Modeling meeting with Rhea and Anshuman to go over details of SimBio for Lotka Volterra Model + making plan for next steps

    • Second Biolink Depot visit

    • Made NEB DH5a mCherry integrants competent (Stephen, David).

    • Gel electrophoresis of SDM products (Tobin)

    • Meeting with a digital PCR specialist to discuss IISER-K wet lab collaboration (Tanya, Julia)

    • Transformed BL21 cells with mCherry & pE-FLP and DH5a with pE-FLP (Rose, Tanya, Julia)

    • Meeting with IISER-K to discuss wet lab plan (Tanya, Julia)

    • Performed Colony PCR of pMINT CRISPR region (Stephen, Tobin)

  • Week of Aug 22 - 28

    • Made liquid cultures of plain DH5a, DH5a-mCherry, DH5a-mCherry-FLP, plain WM3064, and WM3064-mCherry. We looked at the DH5a under the microscope, and the fluorescence was present! (Torrey)

    • Restriction enzyme digest of pSPAIN with BamHI-HF (Nabil, Franklin, Fonz)

    • Colony PCR on pSPAIN mCherry guides 2, 4, & 5 and DH5α-mCherry/pE-FLP. Colony PCR results ran on a gel, DH5a flipped and unflipped bands at expected sizes, successful transformation. Nanodropped samples. (Stephen, Tara, Yi-Chi)

    • Preliminary plate reader and flow cytometry analysis of DH5a-mCherry, WM3064-mCherry, and DH5a non-fluorescent cells showed higher fluorescence for mCherry cells (Tara, Tobin).

    • Design chalk talk on making pMADRID from pSPAIN (Whole team)

    • Made mCherry-pE-FLP- DH5a cell chemicompetent (Rose, Julia)

    • Made glycerol stock of DH5α-mCherry/pE-FLP (Tara)

    • Performed preliminary flow cytometry gating strategy → showed DH5a mCherry fluorescence higher than the non-fluorescent control (Tanya, Tobin)

    • Colony PCR on pOSIP mCherry, and pMINT (guide 2, 4, 5) (Stephen, Tara, Yi-Chi)

    • Midi prepped pSPAIN (Emily, Nabil).

    • Attempted to isolate M13K07 helper plasmid from the M13 phage by infecting WM3064 with M13KO7 phage (Torrey, Tara, Rhea)

    • Continued to attempt to infect WM3064 with M13KO7 (Torrey)

  • Week of Aug 29 - Sept 4

    • Continued to attempt to infect WM3064 with M13KO7 (Torrey)

    • Miniprepped pSPIN (Tobin, David, Denise)

    • Ran gel electrophoresis of pSPAIN midi prep and pSPIN mini prep (Emily, Nabil)

    • Attempted PCR of pSPIN CRISPR region (David, Rose, Denise)

    • Continued to attempt to infect WM3064 with M13KO7 (Torrey)

    • Continued to attempt to infect WM3064 with M13KO7 (Torrey, Tanya, Rhea)

    • Hydrated 50 primers used for pSPAIN sequencing (Emily)

    • Ran PCR on pSPIN and pSPAIN after restriction enzyme digest (Emily, Nabil)

    • Attempted pMADRID Restriction Cloning (2 group pSPAIN, pSPIN digestion) (David, Julia, Tara)

    • Ran PCR of pSPIN CRISPR array (David, Tara)

    • Continued to attempt to infect WM3064 with M13KO7 (Torrey, Tanya, Rhea)

    • Acquired Tet/Cam plasmid pACYC184, grew up cultures (Torrey)

    • pMADRID construction with BamH1 and Sal1 (David, Julia)

    • Educational Outreach at Scotts Valley High School (Rose, Yi-Chi, Rhea, Tara, and Franklin)

    • Attempted inverse PCR of pSPAIN (Emily, Nabil)

    • Miniprepped Tet-Cam Plasmid (Emily, Nabil)

    • Tested Tet plate concentration (Stephen)

    • Continued to attempt to infect WM3064 with M13KO7 (Torrey, Tanya)

    • Continued to attempt to infect WM3064 with M13KO7… got growth on 15ug/mL Kan plate that was thought to maybe be successful but liquid culture still did not grow (Torrey, Tanya)

  • Week of Sept 5 - 11

    • Continued designing IISER-K we lat experiments (Tanya)

    • Attempted to redo of inverse PCR of pSPAIN (Emily and Nabil)

    • Attempted gel of Tet-Cam plasmid (Emily and Nabil)

    • pMADRID miniprep, colony PCR, and plasmid gel electrophoresis (David, Tara, Yi-Chi, Tobin)

    • Prepared overnight culture of WM3064 that we thought might be infected with M13KO7 (Torrey, Tanya, Rhea)

    • Attempted to transform pET-52b(+) into DH5alpha cells and plated. (Franklin, Rose)

    • Attempted to miniprep M13KO7 helper phage out of WM3064 (Torrey, Tanya, Tara, Rhea)

    • First attempt of Golden Gate Assembly to make pMADRID into pMINT (David, Tobin, Yi-Chi)

    • Chemicomp transformation of Pet52b into DH5a

    • Made liquid cultures of pET-52-ccdB colonies 8,9,10 (Franklin)

    • Second attempt Golden Gate Assembly to make pMADRID into pMINT with buffer exchange, only showed growth on positive control (David, Tobin, Yi-Chi)

    • Redid transformation of pET-52b(+) into DH5alpha

    • Ran gel ectrophoresis of miniprep product from WM3064 supposedly infected with M13K07 helper phage (Torrey, Tara)

    • Attempted gel extraction of M13K07 helper phage plasmid (Torrey, Tara, Rhea)

    • Initiated contact with Thermo Fisher customer support to troubleshoot the continued failure to infect WM3064 with M13KO7 helper phage (Torrey)

    • Third attempt of Golden Gate Assembly with 2 hour incubation to make pMADRID into pMINT (David, Tobin, Yi-Chi)

    • Designed and ordered primers to amplify ccdB from pOSIP-KC

    • Made stock of C41 chemicompetent cells (Nabil, Fonz)

    • Thermo Fisher phage specialist emailed to say that our phage issues probably occurred from an incompatibility between our WM3064 cells and M13KO7. We ordered a confirmed compatible strain E. coli DH5a F’ (Torrey)

    • Designed gene blocks for IISER-K wet lab collab (Tanya, Franklin)

  • Week of Sept 12 - 18

    • Meeting with IISER-K to discuss wet lab plan (Tanya, Franklin)

    • Started overnight culture of DH5a with PET-52b(+). (Rose)

    • Educational Outreach → Kaohsiung American School (Rhea, Tanya, Rose, Yi-Chi, Tara)

    • IISER-K collab gene blocks finalized and ordered

    • Fourth attempt of Golden Gate Assembly with dephosphorylation to make pMADRID into pMINT (David, Tobin, Yi-Chi) → fail

    • Attempted to miniprep of pET-52b(+) & pSPAIN (Rose, Tanya, Wen)

    • Titered M13KO7 helper phage stock in E. coli DH5a F’. Could see a difference between colonies and infected plaques, confirming that M13KO7 can infect the new cell line (Torrey, Rhea)

    • Miniprepped and Digested pET-52b, in order to restriction clone the ccdB fragment into the backbone (Rose, Julia)

    • IDT rejected our IISER-K collab gene blocks due to high complexity, had to fix and order again (Tanya)

    • Attempted to PCR amplify ccdB fragment out of pOSIP-KL two times (Tanya, Franklin)

    • Midiprepped pMADRID (Tobin, Nabil)

    • Attempted gel extraction of digested pMADRID (Tobin, David, Torrey, Yi-Chi)

    • Meeting with Jason, Ricardo, and Ethan of UCD iGEM to help them incorporate vaccination into their SIR model (Torrey)

    • Miniprepped pOSIP-KC (Rose)

    • Contacted Dr. Manuel Ares and Dr. William G. Scott (UCSC) for advice on how to synthetically make the molecule that activates Csm6 [the IISER-K protein] (Tanya, Franklin)

    • Attempted one last time to PCR amplify ccdB fragment out of pOSIP-KL. Found out the forward primer caused the failures (Tanya, Franklin)

    • Meeting with IISER-K team to discuss wet lab plan (Tanya)

    • Infected new culture of DH5a F’ with M13KO7 to eventually extract the helper phage plasmid (Torrey, Rhea)

  • Week of Sept 19 - 25

    • Miniprepped the M13KO7-infected DH5a F’ cells. Miniprep product was expected to have the desired M13KO7 plasmid and undesired parts or whole of the F’ episome (Torrey, Rhea)

    • Troubleshoot pSPAIN inverse PCR with gradient temperatures (Stephen, Tobin)

    • Attempted to PCR amplify ccdB out of pOSIP-KC using DMSO and touchdown PCR. (Franklin, Rose)

    • Designed new forward primer for ccdB amplification from pOSIP-KC (Franklin, David)

    • Ran gel electrophoresis of miniprep of M13KO7-infected DH5a F’. Showed that M13KO7 plasmid was there, but showed other unexpected larger and smaller products (Torrey)

    • Fifth attempt at Golden Gate Assembly using new insert oligo duplexes to make pMADRID into pMINT (David, Torrey, Yi-Chi)

    • First attempted transformation of F’ DH5alpha-pOSIP-mCherry with pE-FLP (Emily)

    • Transformed miniprep product of M13KO7-infected DH5a F’ into chemicomp DH5a and selected with Kanamycin. Assumption is that the only cells that would grow would have only M13KO7 plasmid (Torrey)

    • Miniprepped the DH5a transformed with M13KO7 plasmid and unwanted junk (Tobin, Torrey). Gel electrophoresis of that miniprep product showed a faint band where M13KO7 is expected but a bright “band” at top of well. M13KO7 plasmid was likely in that product, but may have been present with other plasmid or chromosomal DNA as well.

    • Discovered the IISER-K gene blocks we ordered were flawed, so we designed and ordered primers to use PCR to fix the mistake (Franklin, Tanya)

    • Followed advice from Dr. William Scott to design a way to produce the molecule to activate Csm6 (Tanya)

    • Ran Colony PCR of various potential pMINT colonies and gel electrophoresis. Miniprepped pMINT plasmid from colonies with successful PCR (David, Yi-Chi, Torrey)

    • Initial colony PCR on potentially flipped DH5alpha F’ mCherry integrants (Denise, Emily)

  • Week of Sept 26 - Oct 2

    • Meeting with Dr. Bruce Levin (Emory University) about the dynamics of plasmid transfer (Torrey, Rhea, Tara)

    • Transformed pET-52 and CAHS1 plasmids into C41 to test competency of cells and compatibility of pET-52 with pLys (native C41 plasmid) - confirmed competency and compatibility (Tara, Rhea)

    • Attempted to transform pMINT guides (2.9, 3.7) and pMADRID (non-targeting) into DH5α-mCherry pE-FLP for use in plate reader experiments

    • Added Kanamycin to M13KO7-infected cultures to select for infectants (Torrey)

    • Sent out pMINT for Sanger sequence (Tobin, Nabil)

    • Successful ccdB amplification from pOSIP-KL with the new forward primer! (Tanya, Franklin)

    • Attempted another ccdB amplification to get more product,PCR failed twice due to human error (Rose, Franklin, Tanya)

    • IISER-K gene blocks arrived. Rehydrated them and attempted PCR to fix mistakes in the sequences—PCR failed (Tanya, Franklin)

    • Meeting with IISER-K to discuss wet lab plan (Tanya)

    • Another successful ccdB amplification from pOSIP-KL after fixing human errors from previous attempts (Franklin)

    • Troubleshooted failed PCR for the IISER-K gene blocks and ran a new-and-improved reaction that was successful (Tanya, Franklin)

    • Performed DH5a F' colony PCR with pE-FLP and attb primers (Rhea)

    • Ran gel on colony PCR → no bands present, no flipped integrants (Denise, Emily)

    • Second attempt at colony PCR using new growth from DH5alpha F’ mCherry pE-FLP Amp plates (Denise, Emily)

    • Miniprepped DH5α -pMINT2.9/3.7, ran gel on miniprep product (Torrey, Tara)

    • Inverse PCR on pSPAIN with new primers. Determined that the best primer combinations were New Reverse Primer 1 + Old Forward with OriT and New Reverse Primer 1 + Old Forward Primer without OriT (David, Torrey)

    • Restriction digest of IISER-K gene block (Tanya, Rose, David)

    • Ligation of pet-52 ccdB (Rose, Tanya)

    • Ligation of pet-52 and IISER-K gene (Rose, Tanya)

  • Week of Oct 3 - 9

    • Completed ligation experiments of pet-52 ccdB and transformation of pet-52-ccdB into DH5aF’ + plating (Franklin, Tanya, Rhea, Fonz)

    • Completed ligation experiments of pet-52 IISER-K gene and transformation of pet-52-csm6 into C41 + plating (Franklin, Tanya, Rhea, Fonz)

    • Grew overnight liquid culture of DH5alpha-mCherry pE-FLP (Rhea, David)

    • Grew overnight liquid cultures of DH5alpha, DH5alpha-mCherry F’, and DH5alpha-mCherry pE-FLP (F’) cells for fluorescence analysis (Emily, Fonz)

    • Colony PCR and Index plate of DH5alpha-F’ that was transformed with pET52-Express. (Julia, Rose, Tanya)

    • Colony PCR of ATTB sites for DH5a-mCherry pE-FLP (Rhea, Yi-Chi)

    • Ran gel for Colony PCR of ATTB sites for DH5a-mCherry pE-FLP (Yi-Chi)

    • Made DH5a-mCherry pE-FLP cells chemicompetent (Tobin, Fonz)

    • Colony PCR to verify successful transformation of pET-52-Csm6 in C41 cells (Tanya, Franklin, Tara, Julia)

    • Ran gel electrophoresis to visualize colony PCR results of pET-52-Csm6 in C41 cells—showed successful cloning and transformation! (Tanya, Franklin, Emily)

    • Successfully transformed pMINT guides (2.9, 3.7) and pMADRID (non-targeting)into DH5α-mCherry pE-FLP for use in plate reader experiment (Tara, Tobin, Yi-Chi)

    • Ran Colony PCR of pET-52-csm6, ccdB; liquid culture (Franklin, Tanya)

    • Preliminary plate reader analysis confirmed that mCherry and pE-FLP integrants in DH5alpha F’ have higher fluorescence than DH5alpha, implying successful transformation (Emily, Tobin)

    • Streaked LB/Amp and LB/Kan plates with liquid culture to grow up for colony PCR (Emily)

    • Started liquid cultures from pMINT (2.9, 3.7) and pMADRID (NT) transformant colonies for plate reader analysis (Tanya, Yi-Chi)

    • Induced protein expression using IPTG for IISER-K wet lab work (Tanya)

    • Attemoed to transform pET-52-Csm6 into DH5a—failed (Tanya, Franklin)

    • Plate reader analysis of pMINT (2.9, 3.7) and pMADRID (NT) transformants suggested successful knockout of mCherry gene function (Tobin, Tara, Rhea, Yi-Chi)

    • Colonies grew on Amp and not Kan. Ran colony PCR using attB primers (Emily)

    • Ran a gel on attB primer PCR products. Band for DH5alpha-mCherry pE-FLP at the expected length, confirming that pE-FLP was successful (Emily)

    • Miniprep of colonies 8, 9, and 10 from DH5alpha-F’ pET52-Express index plate. (Rose)

    • Touchdown PCR with ccdB primers and Gel of miniprep 8, 9, and 10 from DH5alpha-F’ pET52-Express verified that ccdB region was ligated. (Rose, Tanya, Franklin)

    • Inverse PCR on pMINT with new primer combinations to get backbone. Golden gate assembly of phiMINT and phiMINTO. Transformation of phiMINT and phiMINTO into DH5a (David, Torrey).

    • Harvested cell pellet of IPTG-induced cells for protein purification for IISER-K collab (Franklin)

  • Week of Oct 10 - 16

    • Miniprepped DH5a transformed with phiMINT and phiMINTO (David, Julia)

    • Meet with IISER-K iGEM to wrap up modeling (Torrey, Stephen, Rhea)

    • Flow cytometry to test for cell integration (Stephen, Tobin, Tanya)

    • Miniprep of C41 cells with the Csm6-expressing plasmid and attempted gel extraction (Tanya, David)

  • Week of Oct 17 - 21

    • Meet with IISER-K iGEM wet lab meeting (Tanya, Julia)

    • PCR amplification of Csm6 from the miniprep done on 10/14 and gel showing that Csm6 is in fact present in the cells (Tanya, Julia)

    • Csm6 protein purification (Tanya, David, Julia, Yi-Chi)