Jordan

• Helped with Western blot in the morning
• Looked into alternative periplasm fraction options
• Transformed Golden Gates and Gibsons
• 1 uL of Patrick’s transformation control on Amp
• 1 uL of Gibson YcdO product on Cam
• 2 uL “ “ “
• 3 uL “ “ “
• 4 uL “ “ “
• 3 ul of positive Golden Gate control from Patrick on Amp
• 3 ul GG control w/ no ligase on Amp
• 3 uL GG control w/ no insert on Amp
• 3 uL gRNA-guide GG on Tet
• 3 uL GFP-mcherry Golden Gate on Cam
• Used 950 uL of SOC and plated 2 plates of each
• Incubated for 1 hr. 15 minutes

Michelle

• Ran gel on Cas9-SS Gibsons (2)
• Completely empty
• Made 40 fmol/uL dilutions of gRNA template, mCherry1, and Tet lnrz for GFP for GG Golden Gate assembly
• Ran Golden Gate reactions
• mCherry:
• 1 uL of 40 fmol/uL mCherry1 PCR product
• 1 uL of 40 fmol/uL Tet lrnz for GFP PCR product
• 1 uL of 40 fmol/uL TorA
• 2 uL of 10X T4 ligase buffer
• 2.24 uL of 110uL BSA + 10uL T4 ligase buffer
• 0.5 uL of 400,000U/mL T4 ligase from NEB
• 0.5 uL of BsaI
• Water to 21.76 uL
• gRNA:
• 1 uL of 40 fmol/uL gRNA template
• 1 uL of annealed cutting guide insert from primers (straight from tube: ~ 6 uM)
• 2 uL of 10X T4 ligase buffer
• 2.24 uL of 110uL BSA + 10uL T4 ligase buffer
• 0.5 uL of 400,000U/mL T4 ligase from NEB
• 0.5 uL of BsaI
• Water to 21.76 uL
• Positive control:
• 1 uL of Patrick’s positive control “32, 50ng”
• 1 uL of Patrick’s positive control
• 2 uL of 10X T4 ligase buffer
• 2.24 uL of 110uL BSA + 10uL T4 ligase buffer
• 0.5 uL of 400,000U/mL T4 ligase from NEB
• 0.5 uL of BsaI
• Water to 21.76 uL
• Negative control (no ligase):
• 1 uL of Patrick’s positive control “32, 50ng”
• 1 uL of Patrick’s positive control
• 2 uL of 10X T4 ligase buffer
• 2.24 uL of 110uL BSA + 10uL T4 ligase buffer
• 0.5 uL of BsaI
• Water to 21.76 uL
• Negative control (no insert):
• 1 uL of Patrick’s positive control “32, 50ng”
• 2 uL of 10X T4 ligase buffer
• 2.24 uL of 110uL BSA + 10uL T4 ligase buffer
• 0.5 uL of 400,000U/mL T4 ligase from NEB
• 0.5 uL of BsaI
• Water to 21.76 uL
• Conditions:
• 30 cycles, 37°C (2:00) 16°C (2:00) 50°C (5:00) 80°C (5:00)
• In the future add 2 uL of 10X BSA in every reaction tube

Paul

• Finished Western Blot procedure with Ben
• TetR plasmid expressed less Cas9 than Top10
• Fraction procedure may need revision:
• Slight Cas9 residue in periplasm fraction
• Lanes:
• 1: ladder
• 2: Whole cell ClyA-GFP
• 3: Whole cell Neg control
• 4: Whole cell Cas9 in pSB1C3 (Tet promoter)
• 5: periplasm ClyA-GFP
• 6: periplasm Neg control
• 7: periplasm Cas9 in pSB1C3 (Tet promoter)
• 8: ladder
• Phosphorylate and anneal gRNA primers:
• 1 uL PNK
• 0.61 uL of 100 uM primer 1
• 0.61 uL of 100 uM primer 2
• 2 uL T4 ligase buffer
• Water to 20 uL
• @ 37°C for 30 min @ 95°C for 5 min
• Cool to 28 C at ~1°C/min

Sam

• Reached out to museums
• Put finishing touches on pamphlet
• Began writing human practices section for website
• Began writing collaboration section for website
• Emailed Australia

Sara

• Read through the Australians’ protocols and made a list of questions and things that we would need to do them
• Did a little lab notebook work
• Plated the gibson Ycdo and Cas9 gibson products
• 500 uL of the 1000 uL total solutions onto 2 plates each

Tyler

• Mathematical modeling, looked into and researched other antibiotic resistant targets
• Gibson Reaction (Ycdo)
• 2.78 n.f. H₂0
• 0.22 µL Ycdo Insert
• 2 µL (36 ng) SS Lnrz Cas9
• 5 µL Gibson Mix