Team:Penn/Notebook

June 4 2013 - June 11 2013

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

8-Jun

1. Miniprep Addgene stuff + transformations that worked
2. Growing up low copy plasmids in 40mLs

Mini-prepped

1. I9002
2. I13458
3. C0051
4. Pdawn-mcherry
5. Pdawn
6. Dhsa mcherry
7. Pdawn dhsa
8. Psb1a3
9. JM mcherry
10. Pet26b

June 11 2013 - June 18 2013

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

June 18 2013 - June 25 2013

21-Jun

1. 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
11. troubleshoot t9002 digest
1. check for contamination of something (run uncut sample, sample + buffer, sample + 1 enzyme, sample + other enzyme, sample + both enzymes)

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

June 25 2013 - July 2 2013

• 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


• 1-Jul
  1. Beautiful Brady Bunch photoshoot
  2. Troubleshooted and Re-tred PCR for user ends for reporter plasmid
  3. Called IDT about pcr assembly – they said Gibson tends to work better, no mutations, all in one tub. If we must PCR assembly – add DMSO, hotstart reaction, anneal at 68-70C (we did this).
  4. Get methylated biobrick sequenced
  5. Only sequence ptet GFP 11, if verified make sure to note on LIMS that we are only using ptet GFP 11
  6. Check if plux/luxI system is working in liquid cultures – this failed
  1. a. Might be strain competition, need to know growth rates
  7. Re-suspend primers for lux amplifier
  8. Mini-prep: e0040, psb1a3, r0062


• 2-Jul
  
  
  1. Think about application of mathylation project in e.coli
  2. Ceck if plux/GFP-psb1C3 system is working in liquid cultures
     1. +/- AHL induction at 100nM
     2. Compare with ptetGFP fluorescence, normal LB fluorescence
  3. Streak zinc finger 2
  4. Grow up 44251
  5. Transform up R0062
  6. When BstuI arrives
     1. Assay BstuI working
     2. Assay the bvluc and tale1 minipreps in dh5a, dam-, and bl21 cells with msssi and bstuI
     3. Results: BstUI is blocked by methylation, and cells don’t normally methylate
  7. Growing up t9002 in chlor and i751250 in amp for fluorescence study
  8. Investigate CHIP or other ways of determining DNA binding domain specificity

July 2 2013 - July 9 2013

• 3-Jul
  1. Streak zinc finger 2
  2. Grow up 44251
  3. Look into lux box being light sensitive
• 4-Jul
  1. Mini prep 44251
  2. Pick ZFP2 colonies to grow up, throw out liquid culture in fridge
• 5-Jul
  1. Repeat BstUI assay, taking into account new controls
  2. Suspend the primers in the freezer
  3. We need to check if the origin of replications are compatible before co transformation
  4. Characterize pDawn-mcherry
  5. Practice measuring fluorescence
• 6-Jul
  1. troubleshoot ptetGFP user PCR - band was visible but too small to extract
  2. gel extract promoter fragments from USER PCR
  3. re-do USER PCR for: TetR, pTetGFP
  4. Design/check/order (Tale,Cas,ZFP)-flex-REV primers - check fwd primers
• 8-Jul
  1. pick up minigenes and primers from the cell center
  2. pick many colonies, colony PCR, and run results on a gel
  3. Restriction digest sgRNA with RFP for cotransformation with cas9
  4. Find/make/buy TBE for use in TBE gels (hi-resolution)
  5. PCR assemble MsssI with USER primers
  6. get pTetGfp from pcr box and run gel
  7. PCR purify pTetGFP in eppendorf and LIMs it
  8. Fab device(s) and begin fiddling with them
  9. get t9002 seq
  10. pcr luxI, gel verify, pcr purify, restriction digest, ligate into pDawn (remember NIC), transform
  11. redo fluorescence experiment using the new cultures as well - report fluorescence per OD. do it in replicate, also use minimal media