Tuesday, August 23rd
Tasks:
Jordan
- Did more research into periplasm fractioning
- In order to see if the protocol works, need JC8031 with ClyA culture, a JC8031 DeLisa cell line control (same cells, no GFP) and a cytoplasmic GFP control (if there is GFP in the fraction, full cell lysis happened and that’s bad)
- Plan:
- Grow up overnight culture of GFP device from interlab kit
- Grow all three cultures in 25 mL LB cultures with Cam until OD=0.5 per DeLisa paper
- Induce JC8301 cells with .2% arabinose (weight/final volume)
- Grow for 6 hours at 37
- Use cold osmotic shock protocol from Biefeld iGEM team on the drive, BUT use ice cold water instead of cell fraction buffer 2 because Kelly says Triton and SDS can interfere with protein function
- Measure fractions on plate reader, use wavelength from interlab study
- Need: to make cell fraction buffer (need Tris) autoclave media?, grow up cytoplasm GFP, did Paul make some arabinose?
- Made some 3M sodium acetate solution to try ethanol precipitation—Kelly says it’s worth a try—see Paul for final pH
- Ethanol precipitation protocol
Paul
- Looked into Cas9 expression cell lines with Kelly
- Cas9 should be constitutively expressed in any line without TetR repressor (so our cell line)
- Gibson assemble gRNA into mRFP reporter plasmid pSB1T3
- Used Bradley’s Clone Kit Excel calculator
- Pos. Gibson Control
- Neg Gibson Control (no insert)
- 10 uL rxns:
- 4.15 uL nfH20
- 0.41 uL 63 ng/uL mRFP backbone
- 0.45 uL 10 ng/uL gRNA gBlock insert
- 5 uL Gibson Mastermix
- Note for 8/24: L-arabinose 0.2% final w/v for induction of ClyA GFP
Shu
- Miniprepped mRFP, sfGFP, mCherry and nanodropped them
Tasfia
- Gel extraction of GFP1 for GG PCR product from 8.22.2016
- It turned out there was nothing in the ~1000 bp “bands,” and concentrations were to small to get accurate reads (reading negative values)
- Extracted the ~400 bp bands for the sake of gel extraction troubleshooting, and the results were pretty but it wasn’t for the product we wanted
- Concentration: 83.1 ng/uL, 260/280: 1.94, 260/230: 2.03
- Gel extraction of Cas9-Lrz-SS
- Gel pieces were excised and stored in -20°C freezer for two hours
- Mass Excised:
- Lower band 1.25 ng template: 0.1152 g
- High band 1.25 ng template: 0.0405 g
- Lower band 2.5 ng template: 0.1492 g
- Results:
- Lower band 1.25 ng template: 6.9 ng/uL, 260/280: 1.94, 260/230: 2.03
- High band 1.25 ng template: 11.5 ng/uL, 260/280: 2.01, 260/230: 0.73
- Lower band 2.5 ng template: 4.5 g/uL, 260/280: 4.79, 260/230: 0.18
- There are still bands at ~400, where they usually occur, but we seem to have some bands near 1000 bp
- Our gels consistently end up with products that are not where our ladder predicted them to be. From this we can conclude that the amount of template added to the PCR is not causing the bands to appear smaller than they actually are
- Gels screening test:
- Both ladders are inaccurate
- Talked to Emma from Auburn Gresham High
Tyler
- Ran gel on previous day Lnrz Cas9 PCR
- Transformed Gibson Reaction twice (first time on 2 were on wrong plates)
Jordan
- Did more research into periplasm fractioning
- In order to see if the protocol works, need JC8031 with ClyA culture, a JC8031 DeLisa cell line control (same cells, no GFP) and a cytoplasmic GFP control (if there is GFP in the fraction, full cell lysis happened and that’s bad)
- Plan:
- Grow up overnight culture of GFP device from interlab kit
- Grow all three cultures in 25 mL LB cultures with Cam until OD=0.5 per DeLisa paper
- Induce JC8301 cells with .2% arabinose (weight/final volume)
- Grow for 6 hours at 37
- Use cold osmotic shock protocol from Biefeld iGEM team on the drive, BUT use ice cold water instead of cell fraction buffer 2 because Kelly says Triton and SDS can interfere with protein function
- Measure fractions on plate reader, use wavelength from interlab study
- Need: to make cell fraction buffer (need Tris) autoclave media?, grow up cytoplasm GFP, did Paul make some arabinose?
- Made some 3M sodium acetate solution to try ethanol precipitation—Kelly says it’s worth a try—see Paul for final pH
- Ethanol precipitation protocol
Paul
- Looked into Cas9 expression cell lines with Kelly
- Cas9 should be constitutively expressed in any line without TetR repressor (so our cell line)
- Gibson assemble gRNA into mRFP reporter plasmid pSB1T3
- Used Bradley’s Clone Kit Excel calculator
- Pos. Gibson Control
- Neg Gibson Control (no insert)
- 10 uL rxns:
- 4.15 uL nfH20
- 0.41 uL 63 ng/uL mRFP backbone
- 0.45 uL 10 ng/uL gRNA gBlock insert
- 5 uL Gibson Mastermix
- Used Bradley’s Clone Kit Excel calculator
- Note for 8/24: L-arabinose 0.2% final w/v for induction of ClyA GFP
Shu
- Miniprepped mRFP, sfGFP, mCherry and nanodropped them
Tasfia
- Gel extraction of GFP1 for GG PCR product from 8.22.2016
- It turned out there was nothing in the ~1000 bp “bands,” and concentrations were to small to get accurate reads (reading negative values)
- Extracted the ~400 bp bands for the sake of gel extraction troubleshooting, and the results were pretty but it wasn’t for the product we wanted
- Concentration: 83.1 ng/uL, 260/280: 1.94, 260/230: 2.03
- Gel extraction of Cas9-Lrz-SS
- Gel pieces were excised and stored in -20°C freezer for two hours
- Mass Excised:
- Lower band 1.25 ng template: 0.1152 g
- High band 1.25 ng template: 0.0405 g
- Lower band 2.5 ng template: 0.1492 g
- Results:
- Lower band 1.25 ng template: 6.9 ng/uL, 260/280: 1.94, 260/230: 2.03
- High band 1.25 ng template: 11.5 ng/uL, 260/280: 2.01, 260/230: 0.73
- Lower band 2.5 ng template: 4.5 g/uL, 260/280: 4.79, 260/230: 0.18
- There are still bands at ~400, where they usually occur, but we seem to have some bands near 1000 bp
- Our gels consistently end up with products that are not where our ladder predicted them to be. From this we can conclude that the amount of template added to the PCR is not causing the bands to appear smaller than they actually are
- Gels screening test:
- Both ladders are inaccurate
- Talked to Emma from Auburn Gresham High
Tyler
- Ran gel on previous day Lnrz Cas9 PCR
- Transformed Gibson Reaction twice (first time on 2 were on wrong plates)
