iGEM 2021 — Team UCSC

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# Team:UCSC/Notebook

[![](https://static.igem.org/mediawiki/2021/4/4c/T--UCSC--main_logo.svg)](https://2021.igem.org/Team%3AUCSC)

[ ]

* Project +
  [Project](https://2021.igem.org/Team%3AUCSC/ProjectLandingPage)
  [ ]
  + [Description](https://2021.igem.org/Team%3AUCSC/Description)
  + Engineering +
    [Engineering](https://2021.igem.org/Team%3AUCSC/Engineering)
    [ ]
    - Design +
      Design
      [ ]
      * [Gene Elimination](https://2021.igem.org/Team%3AUCSC/Gene_Elimination)
      * [Phage Infection](https://2021.igem.org/Team%3AUCSC/Phage_Infection)
      * [Conjugation](https://2021.igem.org/Team%3AUCSC/Conjugation)
    - [Proof of Concept](https://2021.igem.org/Team%3AUCSC/Proof_of_Concept)
    - [Detection](https://2021.igem.org/Team%3AUCSC/Detection)
  + [Results](https://2021.igem.org/Team%3AUCSC/Results)
  + [Model](https://2021.igem.org/Team%3AUCSC/Model)
  + [Contributions](https://2021.igem.org/Team%3AUCSC/Contribution)
  + Implementation +
    [Implementation](https://2021.igem.org/Team%3AUCSC/Implementation)
    [ ]
    - [Future Directions](https://2021.igem.org/Team%3AUCSC/Future_Directions)
  + [Future Lab Directions](https://2021.igem.org/Team%3AUCSC/FutureLabDirections)
* Human Practices +
  [Human Practices](https://2021.igem.org/Team%3AUCSC/HumanPracticesLandingPage)
  [ ]
  + [Integrated](https://2021.igem.org/Team%3AUCSC/Human_Practices)
  + [Collaborations](https://2021.igem.org/Team%3AUCSC/Collaborations)
  + Partnership +
    [Partnership](https://2021.igem.org/Team%3AUCSC/Partnership)
    [ ]
    - [Wet Lab](https://2021.igem.org/Team%3AUCSC/Partnership#wetlab_start)
    - [Dry Lab](https://2021.igem.org/Team%3AUCSC/Partnership-Dry-Lab#drylab_start)
  + [Outreach](https://2021.igem.org/Team%3AUCSC/Communication)
* Journal +
  [Journal](https://2021.igem.org/Team%3AUCSC/JournalLandingPage)
  [ ]
  + [Notebook](https://2021.igem.org/Team%3AUCSC/Notebook)
  + [Protocols](https://2021.igem.org/Team%3AUCSC/Experiments)
  + [Safety](https://2021.igem.org/Team%3AUCSC/Safety)
  + [Medals](https://2021.igem.org/Team%3AUCSC/Medals)
* [Team](https://2021.igem.org/Team%3AUCSC/Attributions)
* [Glossary](https://2021.igem.org/Team%3AUCSC/Glossary)

![pages flipping through book](https://static.igem.org/mediawiki/2021/3/36/T--UCSC--journal_notebook.gif)

###### 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)

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# Team:UCSC/Engineering

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###### Engineering & Design

* Gene Elimination
* Phage Infection
* Conjugation

We started with a vision: eliminating virulent phenotypes without harming the resident bacterial community. To meet this goal, we designed a mechanism to deliver an engineered cassette to a bacterial population, find and disrupt only the target gene, and propagate the machinery to widespread success. We brought this design to reality by engineering a series of plasmids that can do exactly those things in a lab-safe *E. coli* model. We also designed an aptamer-based lateral flow assay for the detection of Shiga Toxin. Eventually, Progenie could be modified for the elimination of the *stx* gene. Click on a tile above to uncover more details.

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# Team:UCSC/Experiments

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![pipet streaking a plate](https://static.igem.org/mediawiki/2021/3/30/T--UCSC--journal_experiments.gif)

###### Protocols

## Plasmid Cloning

* [pMADRID restriction enzyme cloning](https://static.igem.org/mediawiki/2021/8/80/T--UCSC--pMADRID_restriction_enzyme.pdf)
* [pMINT construction](https://static.igem.org/mediawiki/2021/5/56/T--UCSC--pMINT_construction_protocol.pdf)
* [φMINT and φMINTO Golden Gate Assembly](https://static.igem.org/mediawiki/2021/b/b4/T--UCSC--phiMINT_golden_gate.pdf)
* [pET-52-Csm6 plasmid construction](https://static.igem.org/mediawiki/2021/e/ea/T--UCSC--csm6_plasmid_assembly.pdf)
* [pET-52-ccdB plasmid construction](https://static.igem.org/mediawiki/2021/c/cd/T--UCSC--destination_expression_plasmid_assembly.pdf)

## Fluorescence Analysis

* [Plate Reader](https://static.igem.org/mediawiki/2021/e/ef/T--UCSC--plate_reader_protocol.pdf)
* [Flow Cytometry](https://static.igem.org/mediawiki/2021/1/10/T--UCSC--flow_cytometry_protocl.pdf)

## Standard Operating Procedures

* [Colony PCR (Using OneTaq)](https://static.igem.org/mediawiki/2021/9/91/T--UCSC--colony_pcr_protocol.pdf)
* [Chemicompetent Transformation](https://static.igem.org/mediawiki/2021/6/6c/T--UCSC--chemicomp_transformation.pdf)
* [Inoue chemiompetent cell preparation](https://static.igem.org/mediawiki/2021/8/8a/T--UCSC--Inoue_Chemicomp_cell_prep.pdf)

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# Team:UCSC/Results

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      * [Phage Infection](https://2021.igem.org/Team%3AUCSC/Phage_Infection)
      * [Conjugation](https://2021.igem.org/Team%3AUCSC/Conjugation)
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![spinning planet](https://static.igem.org/mediawiki/2021/6/62/T--UCSC--project_results_header.gif)

###### Results

## We Created *E. coli* DH5α with chromosomally-integrated *mcherry* to use as a model for STEC.

For our *mcherry* Model design, see our[Proof Of Concept](https://2021.igem.org/Team%3AUCSC/Proof_of_Concept)page.

The chromosomally-integrated *mcherry* model was generated utilizing a Clonetegration protocol [[1](#references)]. NEB5α Competent *E. coli* cells were transformed with pOSIP-KL-mcherry [[2](#references)] to integrate the entire pOSIP cassette into the genome at the lambda *attB* site. We made those transformants chemically competent, then subsequently transformed them with pE-FLP to “flip out”, or excise, the integration cassette containing the kanamycin resistance and phage integrase genes.

We verified successful Clonetegration by streaking colonies on ampicillin and kanamycin plates and observing that pE-FLP transformants lost their resistance to kanamycin. We also performed colony PCR on the chromosomal lambda *attB* site in order to verify successful integration after transformation of pOSIP-KL-mcherry, and excision of the integration cassette after transformation with pE-FLP [[Fig. 1](#fig_1)]. Fluorescence was verified utilizing a plate reader where DH5α non-fluorescent cells were compared to three replicates of non-targeting pMADRID into DH5α-*mcherry* cells [[Fig. 2](#fig_2)].

![](https://static.igem.org/mediawiki/2021/8/8d/T--UCSC--colony_pcr_dh5_mcherry.png)

## Figure 1: A 1% agarose gel depicting colony PCR products of both pOSIP-KL-mcherry integrants and colonies flipped after pE-FLP transformation. The primers used in this colony PCR were designed to amplify the lambda *attB* site on the DH5a chromosome. The expected product when the entire OSIP cassette is integrated is 6.8 kb, and the expected product for flipped integrants is 3.4 kb.

![](https://static.igem.org/mediawiki/2021/0/0e/T--UCSC--plate_reader.png)

## Figure 2: Plot of relative fluorescence units for DH5α and DH5α-*mcherry* integrants measured from plate reader. Error bars represent standard error and data was normalized using OD600. A Mann-Whitney U test was performed between a DH5α non-fluorescent control and DH5α-*mcherry* integrants with p=1.28E-6 (<0.05) indicating that the DH5α-*mcherry* integrants are significantly more fluorescent than the non-fluorescent control. Analysis performed in R and figures made with Adobe Illustrator.

## Assembled our destination vector pMADRID

The BamHI-DR-BSAI-gRNA-BSAI-DR-SalI region was PCR amplified from pSL1142 (pSPIN) plasmid [[3](#references)], treated with DpnI, and cleaned using a Monarch PCR clean up kit [[Fig. 3](fig_3)]. The PCR product containing the gRNA and pSL1812 (pSPAIN, generously donated by Leo Vo and the Sternberg lab at Columbia university) were double digested with BamHI and SalI. The vector pSPAIN was incubated with TSAP to dephosphorylate both the backbone and the excised DNA, then ligated with the PCR product from pSPIN. The ligation product was transformed into NEB 5-alpha Competent *E. coli.* Colonies growing on kanamycin with this new construct (pMADRID) were screened using colony PCR [[Fig. 4](#fig_4)]. Positive samples were grown in liquid cultures and the plasmid was isolated in order to use Sanger sequencing to verify successful cloning [[Fig. 5](#fig_5)].

![](https://static.igem.org/mediawiki/2021/6/6f/T--UCSC--pcr_pmadrid_crispr.png)

## Figure 3: A 2% agarose gel depicting the expected PCR product from the pSPIN BamHI-DR-BSAI-gRNA-BSAI-DR-SalI region. The bands are the expected 227 bp length, and these fragments were used as the insert to assemble the pMADRID construct from pSPAIN.

![](https://static.igem.org/mediawiki/2021/2/29/T--UCSC--pMADRID_construct.png)

## Figure 4: A 2% agarose gel showing the colony PCR products from pMADRID assembly transformant colonies. With primers that bind to the pMADRID-specific spacer, the presence of a band indicates the presence of a properly assembled pMADRID construct. We miniprepped six positive samples and verified successful cloning with Sanger sequencing.

![](https://static.igem.org/mediawiki/2021/3/33/T--UCSC--figure_6_results.png)

## Figure 5: A screenshot of Sanger sequencing reads (forward and reverse) for the pMADRID CRISPR array. This image is zoomed into the CRISPR array to show that the 32 nt. spacer was successfully replaced with the expected BsaI placeholder sequence. The rest of the reads agreed with the reference sequence. Figure made with Geneious Prime.

## Assembled our novel vector pMINT that carries an *mcherry*-targeting CRISPR guide

We made three attempts at cloning into pMADRID’s BsaI spacer using Golden Gate assembly, however we were unsuccessful. In our first attempt, we digested pMADRID and *mcherry*-targeting spacers (32 bp) with BsaI and ligated the fragments in a one-pot reaction with a one-insert incubation protocol. The assembly mix was transformed into homemade chemi-competent DH5α cells and no transformants grew. Our second attempt involved a cycling incubation protocol that favors digestion and ligation in separate cycles (37℃ for digestion and 16℃ for ligation), however no transformants were observed.

Finally, we successfully assembled *mcherry*-targeting by substituting our BsaI gBlocks with hybridized oligonucleotides that did not require digestion at all, and had no end fragments that got in the way of ligation. We mixed two oligonucleotides with 34 bases of complementarity corresponding to the 32 nt *mcherry*-targeting spacer and two extra bases corresponding to the *V. cholerae* repeat. The oligos included four extra bases on their 5’ ends that were able to anneal to the overhangs on the vector left behind by the BsaI. The oligos in the mixture were phosphorylated with T4 PNK, then heated to 95℃ for 2 mins and allowed to slowly cool at room temperature. These hybridized oligoduplexes were mixed with digested pMADRID and ligated for 30 mins at room temperature. The ligation product was transformed into NEB 5-alpha Competent *E. coli*. Colonies growing on kanamycin with this *mcherry*-targeting construct (pMINT) were screened for using colony PCR. Potentially positive samples were grown in liquid cultures and miniprepped, and successful cloning was verified with Sanger sequencing [[Fig. 6 and 7](#fig_6)].

![](https://static.igem.org/mediawiki/2021/e/ea/T--UCSC--pMINT_guide.png)

## Figure 6: Sanger sequencing data for minipreps from three screened colonies mapped to the reference CRISPR array for guide 2. The six reads below the reference correspond to forward and reverse sequencing reactions for the three preps. They all showed perfect sequence homology, indicating that the cloning worked for guide 2. Sequencing data analyzed with Geneious Prime.

![](https://static.igem.org/mediawiki/2021/2/22/T--UCSC--figure_7_results.png)

## Figure 7: Sanger sequencing data for three potential pMINT constructs mapped to the reference CRISPR array. The two reads below the reference correspond to forward and reverse sequencing reactions for the one potential construct. This construct showed perfect sequence homology, indicating that the cloning worked for guide 3. Sequencing data analyzed with Geneious Prime.

## Functional assay verified the *mcherry* targeting and elimination abilities of pMINT

Sequence-verified pMINT-Guide-2, pMINT-Guide-3, and non-targeting (NT) pMADRID were transformed into our model DH5α-*mcherry* cells and plated on kanamycin for 24 hours. Resulting colonies and DH5α non-fluorescent cells were grown up in liquid culture for 16 hours and analyzed on the Spectramax ID3 plate reader to determine relative fluorescence. Decrease in fluorescence was observed utilizing a plate reader, fluorescence microscopy, and flow cytometry, where DH5α non-fluorescent cells were compared to three replicates of DH5α-*mcherry* cells transformed non-targeting pMADRID, pMINT with guide 2, and pMINT with guide 3.

Figure 8 shows a plot of relative fluorescence units for non-fluorescent cells and cells transformed with pMINT, pMINT guide 2, and pMINT guide 3. We saw significant reduction in fluorescence for our *mcherry*-targeting constructs, indicating that guided-DNA integration into the promoter of a gene successfully knocks out expression. Figure 9 shows pictures taken of DH5α-*mcherry* cells transformed with non-targeting pMADRID and pMINT guide 2 under a microscope. The top two images show the non-targeting transformants under different light conditions, and the bottom two images show the pMINT guide 2 transformants under the same conditions. We see a similar amount of cells in both streaks, but the targeting constructs resulted in a decrease in fluorescence.

![](https://static.igem.org/mediawiki/2021/4/4d/T--UCSC--figure_plate_reader.png)

## Figure 8: Plot of relative fluorescence units for plain DH5α transformed with control plasmid and DH5α-*mcherry* transformed with pMADRID (non-targeting guide), pMINT Guide 2, and pMINT Guide 3 as measured using a plate reader. Error bars represent standard error and data was normalized using OD600. Results indicate transformation of pMINT Guides 2 and 3 decrease fluorescence of DH5α-*mcherry* integrants. A Mann-Whitney U test performed between the DH5α-*mcherry* non-targeting control and pMINT Guide 2 (p = 2.20E-10 < 0.05) and pMINT Guide 3 (p = 1.48E-8 < 0.05), indicate significantly decreased fluorescence. Analysis performed in R and figures made with Adobe Illustrator.

![](https://static.igem.org/mediawiki/2021/6/6a/T--UCSC--figure_9_results.png)

## Figure 9: Microscope imaging of E. coli DH5a-mcherry cells to visualize fluorescence reduction after transformation with pMINT. A) DH5a-mcherry non-targeting transformant colony under fluorescent filter. B) DH5a-mcherry colony 1 under dark filter. C) DH5a-mcherry-MINT colony 2 under fluorescent filter. D) DH5a-mcherry-MINT colony 2 under dark filter. Panels B and D show that the two different populations have similar cell density, and panels A and C show that the pMINT-transformed cells have decreased fluorescence. Photos were taken with a Nikon Z6 camera with 40x magnification, 1/20s, and ISO 300.

![](https://static.igem.org/mediawiki/2021/e/e1/T--UCSC--flow_cytometry_visuals.png)

## Figure 10: Representative flow cytometry gating strategy for determination of cell fluorescence.

![](https://static.igem.org/mediawiki/2021/2/2b/T--UCSC--flow_cytometry_pmint.png)

## Figure 11: Plot of percent fluorescent population for plain DH5α transformed with control plasmid and DH5α-*mcherry* transformed with pMADRID (non-targeting guide), pMINT Guide 2, and pMINT Guide 3 as measured by flow cytometer. Error bars represent standard error. Results indicate transformation of pMINT Guides 2 and 3 decrease fluorescence of DH5α-*mcherry* integrants. A Mann-Whitney U test performed between the DH5α-*mcherry* non-targeting control and pMINT Guide 2 (p = 4.11E-5 < 0.05) and pMINT Guide 3 (p = 4.11E-5 < 0.05), indicate significantly decreased fluorescence. Analysis performed in R and figures made with Adobe Illustrator.

## Assembled the phagemid vector φMINT and mobilizable vector φMINTO

After verifying our pMINT constructs and testing them against our *mcherry* model cells, we used Golden Gate assembly to build two phagemid plasmids. Using around-the-horn PCR with primers containing BsaI sites on their 5’ ends, we amplified the INTEGRATE gene cassette from pMINT for use as a backbone in Golden Gate assembly.

The first phagemid, termed φMINT, was assembled using an f1 ori gBlock for M13 phage packaging and a chloramphenicol resistance gene (CmR) to replace pMINT’s kanamycin resistance gene. The second phagemid, termed φMINTO, was assembled using an f1 ori, CmR, and an oriT specific to the F’ DH5α strain from NEB. The addition of an oriT should allow the phagemid to be conjugated by cells infected by the M13 phages.

These phagemids were assembled using a one-pot Golden Gate reaction with BsaI and a cycling protocol that cycled between 37C and 16C. The assembly was transformed into chemically competent DH5α and transformants were plated on chloramphenicol plates to select against unmodified plasmids. Transformants growing on chloramphenicol were grown up, miniprepped, and analyzed with gel electrophoresis [[Fig. 12](#fig_12)]. In the future, we plan to sequence these constructs with NGS.

![](https://static.igem.org/mediawiki/2021/c/ca/T--UCSC--gel_phiMINT.png)

## References:

* [[1] One-Step Cloning and Chromosomal Integration of DNA](https://pubs.acs.org/doi/pdf/10.1021/sb400021j)
* [[2] Addgene pOSIP-KL-*mcherry*](https://www.addgene.org/116947/)
* [[3] Addgene pSL1142 (pSPIN, pBBR1 backbone)](https://www.addgene.org/160730/)

# [Integration](#integration)

# [pMADRID](#pmadrid)

# [pMINT](#pmint)

# [φMINT & φMINTO](#phagemid_vector)

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