Team:Penn/Notebook

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Penn's iGEM Team
  • 4-Jun
    1. Learned how to make competent cells, growing up two strains for tomorrow
    2. Transformed 8 plasmids
    3. Determined EL222 fusion is risky but still going ahead with it
    4. Linkers are totally setlled
    5. Found zinc finger plasmid and updated target sequence
    6. Learned how to make tetr- mcherry fusion
    7. Settled on 5 promoters
  • 5-Jun
    1. Learned how to make competent cells, testing them and then making more tomorrow
    2. Transformed 8 plasmids again
    3. Made primers to clone the TET-GFP reporter system, the mCherry promoter strength system, ready to order
    4. Made ultramers for variable promoter blocks (and no target neg controls) – ready to order
    5. Spilled a lot of iced tea outside, bummer
    6. Started primers for dna binding machines
    7. Got a handle on cas9 fusions (pun intended).
    8. Put awesome pics in dropbox
  • 6-Jun
    1. Clean up dropbox
    2. Update budget sheet with addgene and cell center orders
    3. Finish primers for fusion
    4. Set up plate reader for GFP and mCherry assays
    5. run minipreps on pdawn, pdawn-mcherry, pet26b
    6. Grow up mCherry stock
    7. Wrote Penn iGEM on our plasmid
  • 7-Jun
    1. Transform
      1. C0012 –amp/chlor (do both)
      2. M11307 – amp/chlor (do both)
      3. I13458 – amp/chlor (do both)
      4. R0010 – amp/chlor (do both)
      5. R0051 – amp
      6. K206000 –chlor
    2. Start the LIMS and file all the strains and DNA we have made/ ordered
    3. Mini-preped
      1. I9002
      2. I13458
      3. C0051
      4. Pdawn-mcherry
      5. Pdawn
      6. Pet26b
      7. Dhsa mcherry
      8. Pdawn dhsa
      9. Psb1a3
      10. JM mcherry
  • 8-Jun
    1. Miniprep Addgene stuff + transformations that worked
    2. Growing up low copy plasmids in 40mLs
  • 8-Jun
    1. Miniprep Addgene stuff + transformations that worked
    2. Growing up low copy plasmids in 40mLs
    3. Transformed everything that has failed
  • 17-Jun
    1. Grow up luxI culture and grow up tetR culture
    2. Sequence all of the minipreps
    3. Transform t9002 in psb1A3 in NEB10
    4. Retransform ptetGFP to see if BL21DE3 cells are competent
    5. Transform r0079, k081015, r0063 in NEB10
    6. Miniprep psb1k3
    7. Redo dam gel with more dna
    8. Figure out second control zfp from addgene
    9. Figure out how to add luxR binding site to target region
    10. Order sequencing primers for all addgene minipreps
    11. Bisulfite converted msssi methylated c0051
  • 18-Jun
    1. The bisulfite conversion was missing a negative control (bisulfite converted but unmethylated c0051) we'll need this to interperet results
    2. Miniprep c0078, c0079 + make glycerol stocks check same plasmid with our kit
    3. Order 13420 (second zfp)
    4. Design a way to make variable promoters more easily varied (for a biobrick so teams can use the reporter plasmid for their own nefarious reasons)
  • 19-Jun
    1. Transform failed transformation
    2. Make competent DH5a && Dam-
    3. Figure out methylation assays for promoters
    4. Miniprep psb1A3 && all the 40mL cultures
    5. Picked many colonies
    6. Check pTet-gfp under blue light
  • 20-Jun
    1. run plux-luxI pcr
    2. run pdawn-luxI pcr
    3. run pDawn-tetR pcr
    4. run pet26b-tetR pcr and
    5. run pDawn-GFP pcr
    6. run pDawn-mCherry-secretion tag pcr
    7. Nano drop last nightÕs mini preps to check for accuracy
    8. Culture amp resistant successful transformations
    9. Make 5 L LB
    10. Miniprep all the successful transformations w/ new protocol
  • 21-Jun
    1. Jun troubleshoot plux-luxI pcr
    2. roubleshoot pdawn-luxI pcr
    3. made pDawn-tetR pcr work
    4. troubleshoot pet26b-tetR pcr
    5. troubleshoot pDawn-GFP pcr
    6. troubleshoot pDawn-mCherry-secretion tag pcr
    7. miniprep growing cultures, be sure to pick only the glowing ligations
    8. ransform the correct t9002 amp ligation - determined from gel
    9. digested t9002 in amp and ptet gfp in amp to identify the correct ligation
    10. all of the chosen ptet gfp ligations worked, but let's not use 5 || 13 12. troubleshoot t9002 digest
  • 24-Jun
    1. Dam-/dh5a v dam methylation + dpnI && dpnII digest
    2. Digested/ligated/transformed t9002 in amp
    3. Get methylated biobrick sequenced
    4. get chlor backbones sequenced
    5. culture t9002 transformations in liquid media with i751250
    6. mini prep stuff in the incubator
    7. figure out the primer issues 8
    8. Pick t9002 colonies for miniprep
  • 26-Jun
    1. Dam-/dh5a v dam methylation + dpnI && dpnII digest
    2. Digested/ligated/transformed t9002 in amp
    3. get chlor backbones sequenced
    4. culture t9002 transformations in liquid media with i751250
    5. mini prep stuff in the incubator
    6. figure out the primer issues
    7. Pick t9002 colonies for miniprep
    8. USER Cloning reporter plasmid

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