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Team:Queens Canada/Notebook/Week9
From 2012.igem.org
Notebook - Week 9
| Date | Protocol | People | DNA (if relevant) | Quantities and Parameters (if relevant) | Notes on Protocol/ Gel Lanes |
|---|---|---|---|---|---|
| Jun-24 | Liquid Culture of BL21 | Kevin, Beini | BL21 Cells | ||
| Colony PCR | Kevin, Beini | Mastermix Kapa: 6 uL F primer: 0.75 uL R primer: 0.75 uL dd H2O: 12.5 uL Template DNA: 5 uL Total: 25 uL Program:1. Denaturation (Initial): 95 for 5 mins 2. Denaturation: 98C for 20 seconds 3. Annealing: 55C for 15 seconds 4. Extension: 72 degrees, 35 seconds 5. Repeat step 2-4 for 30 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |||
| Gel | Kevin, Maggie | Gel electrophoeresis of fliC 1 from last night | Gel: 1) Ladder, 2)fliC Control, 3) fliC 1, 4) fliC 2 5) fliC 3, 6) fliC 4, 7) fliC 5, 8) fliC 6 | ||
| Prepped DNA from BL21 liquid culture | BL21 Liquid Cultures | ||||
| Jun-25 | PCR | Victor, Phillip | PCR Mastermix dd H20 10 uL (6 uL) Hotstart 12.5 uL LR 0.75 uL RP 0.75 uL Template 1 uL (5 uL) Total 25 uL Program:1. Denaturation (Initial): 95 for 5 mins 2. Denaturation: 98C for 20 seconds 3. Annealing: 50C for 15 seconds 4. Extension: 72 degrees, 35 seconds 5. Repeat step 2-4 for 30 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | Gel: 1)Ladder, 2) FliC Ctrl, 3) fliC 1, 4) fliC 2, 5) fliC 3 | |
| Liquid Culture | Victor, Phillip | ohba and fcs+RBS | |||
| Jun-26 | Heat shock Transformation | 3 Trials with 2 uL of 0.025 ug/ uL 2nd constant domain plasmid --> Should give 50 ng of plasmid DNA - on ampicillin | |||
| Miniprep | ohbA and fcs+RBS | ||||
| Jun-27 | Liquid Cultures | Faisal, Beini | fliC C2 - Trials 2,3 w/ [A] + [K] | ||
| Jun-28 | Liquid Culture | David, Victor, Dan | fliC liquid culture | ||
| PCR | Mastermix Kapa: 12.5 uL F primer: 0.75 uL R primer: 0.75 uL dd H2O: 9 uL Template DNA: 2 uL Total: 25 uL Program:1. Denaturation (Initial): 95 for 5 mins 2. Denaturation: 98C for 20 seconds 3. Annealing: 55C for 15 seconds 4. Extension: 72 degrees, 30 seconds 5. Repeat step 2-4 for 30 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | Gel: 1) Ladder, 2)fliC Control, 3) fliC 1, 4) fliC 2 | |||
| Genomic DNA isolation | Victor, Beini | BL21 | Refer to Genomic DNA isolation protocol | ||
| Liquid cultures | David, Faisal | fliC C2 | |||
| Jun-29 | PCR | Phillip, Victor | BL-21/ CR Domain | Mastermix Kapa: 12.5 uL F primer: 0.75 uL R primer: 0.75 uL dd H2O: 9 uL Template DNA: 2 uL Total: 25 uL Program:1. Denaturation (Initial): 95 for 5 mins 2. Denaturation: 98C for 20 seconds 3. Annealing: 55C for 15 seconds 4. Extension: 72 degrees, 30 seconds 5. Repeat step 2-4 for 30 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | Gel: 1) Ladder, 2) FliC control (BL-21 --C), 3) fliC 1 (BL-21 1) 4) fliC 2 (BL-21 2), 5) Ladder, 6) XbaI Digestion 7) BL-21 Pure DNA, 8) E/S Digestion, 9) Synthesized DNA Repeat Gel: 1) Ladder, 2) FliC Control (BL-21 C), 3) FliC 1 (BL-21 1), 4) FliC 2 (BL-21 2), 5) XbaI Digest, 6) BL-21 DNA, 7) EcoRI SepI Digestion Flic C2, 8) Flic C2 MPP |
| Digestion | BL-21 w/ XbaI using the standard protocol | ||||
| Liquid Culture | BL-21 |
Monday June 25
In the morning, Phillip and Victor did a PCR and gel of fliC1 using a newly grown liquid culture of BL21 and trying different methods to isolate the template DNA better. The gel still turned out blank. What will it take to see that little white band show up ~700 kb on the dark imposing gel landscape? The rest of us did some research on new primers and zinc finger nucleases. In the evening, the members of SynthetiQ convened. We made a timeline, wish list of concepts we would like to illustrate, and crossed our fingers for an artist-in-residence.
Tuesday June 26
In the morning, we examined a potpourri of research topics that included zinc finger nucleases, converting the E. coli fliC sequence to BB-2 standard, analysis of the S. typhimurium fliC sequence, and genomic DNA isolation protocols. In the lab, David and Victor resuspended the fliC second constant domain we got synthesized, performed heat shock transformation, and grew the transformed bacteria on three ampicillin plates. Kevin had a meeting with Dr. Steven Smith and came back with an exciting new prospect: using cohesins and dockerins in our chimeric flagella. These proteins are part of a multi-enzyme complex called the cellulosome that some anaerobic bacteria use to degrade cellulose. This might just be the perfect application for our flagellar scaffold that we have been chasing after these past two months. In the afternoon, we all plunged right into this research topic. Rather than forge ahead with another haphazard colony PCR on a very limited supply of Hot Start polymerase, we have decided to wait until tomorrow to ask our faculty advisors for their input.
Wednesday June 27
In the morning, we continued researching the cohesin-dockerin complex and made liquid cultures of the fliC C2 domain. We also received some freebies from New England Biolabs, how wonderful. In the afternoon, we had our biweekly meeting with our faculty advisors. They have given us lots of good advice on how to solve our PCR problems and set us on the right path to amplifying the fliC C1 domain, a key threshold of completing our project. Afterwards, we worked on finding gene sequences for cohesins and dockerins.
Thursday June 28
This morning, Dan, David, and Victor went into the lab to perform a PCR of fliC C1 using the first method we tried (which involved boiling the culture without spinning it down), because there was something present in the wells of the first gel electrophoresis. Everyone else worked on finding cohesin/dockerin gene sequences for 12 species that are known to produce cellulosomes. Kevin decided that we should formally split into research sub-groups so that each person is responsible for gathering comprehensive research and data for a certain topic from start to finish. Briefly, the sub-groups are: Fluorescence, Heavy Metals, Adhesion, Catalysis, Flagella Polymerization and Chemotaxis, and Restriction Enzymes. In the afternoon, Kevin and Phillip met with Dr. Dave Zechel to discuss potential enzymatic pathways we could express on our flagellar scaffold. In the lab, David and Faisal mini-prepped our synthesized fliC C2 gene and made liquid cultures of fliC C2 and BL21. Victor and Beini attempted genomic DNA isolation of BL21.
Friday June 29
In the morning, Victor and Phillip performed a digestion of the genomic DNA preparation we did yesterday, and ran it on a gel to prove that we did manage to isolate the genomic DNA of BL21. They also did colony PCR using genomic DNA as the template DNA. However, some ladder leaked into neighbouring wells, so Beini redid the gel in the afternoon using an 8-well comb rather than a 16-well comb. The only thing that showed up on the later gel was our miniprep of fliC C2, but on the first gel we got some bands for the BL21 genomic DNA. Hmmm. The bottom line is, we still have yet to amplify our vital fliC C1 gene from BL21. Throughout the day, we worked on our respective research topics. At lunch, some of us enjoyed free pizza courtesy of the Alma Mater Society, our school’s student government.
