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
  12. Do I751250(in pDAWN) + T9002 (in psb1a3) co-transformation
  13. MoveT9002 into psb1k3 to be compatible with pdawn - first re-transform psb1k3 then digest/ligate/transform/miniprep
• 9-Jul
  1. Make clear media for lux stuff hold on this until device
  2. Run gel to confirm MsssI PCR assembly/ before meeting-then gel extract the correct band
  3. nanodrop pTetGFPUSER/ TetR USER/ promoters (pcr products)
  4. Run gel to confirm tetR/ 4 promoters PCR
  5. Troubleshoot these USER PCR's (msssI,tetR,4 promoters): msssi
  6. Label gel and send out images and analysis (tetr/promoters)
  7. 1st round DBD pcr's add his tag and flex user site
  8. grow up c0040 (TetR) glycerol stock and miniprep and then sequence
  9. redo TetR sequencing of our current miniprep just to be sure
  10. transform C0179 (lasR without LVA degradation tag) in DH5a, pick colonies, miniprep/glycerol stock
  11. miniprep T9002/amp from glyc at H10 and C0079 (grow overnight on Thurs to miniprep at same time as C0179)
  12. Redesign LuxI into pDAWN primers (NdeI, BamHI)

July 10 2013 - July 17 2013

11-July

1. when we have purified pcr USER product of petgfp/tetR/ promoters - USER fuse the reporter plasmids - note: if there is only one band, then you don't even need to pcr purify
2. Trouble shoot 1st round DBD pcr's add his tag and flex user site
3. We've grown up c0040 (TetR): make a glycerol stock and miniprep and then sequence-it grew in the wrong culture, currently waiting for it to regrow
4. redo TetR sequencing of our current miniprep just to be sure
5. redo 1st round DBD pcr's - add his tag and flex site
6. miniprep T9002/amp from glyc at H10 and C0079 (grow overnight on Thurs)
7. Redesign LuxI into pDAWN primers (NdeI, BamHI)
8. use linearized psb1k3, go ahead and do the digest/ligation t9002

13-July

1. add in primers to MsssI PCR
2. redo Step 1 of plan - pcr assembly MsssIwith new primers - pfu. do we have enough msssi to bother with taq?
3. do step 7 simultaneously - ie. take some of Part 1 pot into new reaction pot with (7) primers
4. PCR purify pfu version of 1st round DBD pcr
5. step 3 - 2nd Round dbd PCR
6. Step 5 of plan - PCR pet26b (50ul rxn) (ie. go ahead with zf/tale even though we need to wait up for cas9 backbone)
7. streak lawned user fusions of reporter plasmid, grow in SOC for an hour, dilute and use new plates to get single colonies

14-July

1. trouble shoot gels of step (4) and (3) and (5). figure out which PCR to redo. which to go ahead and digest etc
2. run tae gel with both msssi pcrs, re-run pet26b pcr, re-run cas9rd2, re-run sgRNA1 pcr, run all of TALE1rd2 for gel extraction
3. image/analyze big TAE gel. Gel extract TALE 2.5kb
4. step (5) - redo pet26b PCR with lower annealing temp, troubleshoot more
5. step (3) - redo cas and TALE PCR for cleaner result. troubleshoot somehow.
6. Gel image redo of cas, Tale, sgrna1, and pet26b.
7. step 4 - redo sgRNA1 PCR, maybe lower annealing temp?
8. Digest 44251 with EcoRI and XbaI
9. Gel extract 44251 digest

15-July

1. 3rd try cas9/TALE rd2 and pET26b PCR.
2. salvage/Redo MsssI pcr
3. redo TetR PCR from biobrick and from Mo's new miniprep
4. grow up pet26b glycerol stock for miniprep
5. Write Protocol: Check rep plasmids fluorescence, if there are no NIC colonies: pick 3 colonies of each to grow,
6. Rep Plasmids: take dilutions and see if they fluoresce with tetracycine induction, use this to choose clones for glycerol stock, miniprep, and seq.
7. grow up R0071 colony for miniprep
8. redo the standard curve - measure tomorrow
9. test sender receiver in M9 - growing up, need to induce tomorrow (maybe)
10. redo testing spent media - growing up, need to induce tomorrow
11. troubleshoot kan tranformations - WE CANT LET THE KANAMYCIN WIN!

16-July

1. run Gel of Cas/Tale rd 2 & pET26b
2. Miniprep pet26b then continue with step (4)
3. step4- digest/ligate (NIC)/ transform sgrna1 w/ pet26b. do simultaneously with (7)
4. Check RP's colonies fluorescence/ NIC colonies, pick 5 to grow.
5. Since NIC (no insert control) grew, we need to colony PCR these guys
6. Order internal primers for MsssI
7. Redo Cas9 and Tale rd 2 with PFU turbo cx
8. do we need new reverse primers for Cas9 and Tale rd 2?
9. Figure out western/SDS page to see if fusions are being expressed - send protocol. Note we have his-tagged our fusions, if that helps at all

17-July

1. run pET26b gel
2. continue (4) - colony pcr, grow 3 correct cultures (pet26b+sgrna)
3. Check Rep. Plasmid's fluorescence, pick 3 colonies for colony PCR if NIC is empty, 5 if NIC is small growth, start over if NIC grows well
4. Plan tetracycline induction expt
5. grow up T9002 chlor for miniprep

July 18 2013 - July 24 2013

18-July

1. Redo pET26b USER PCR with pfu
2. Call taq/Pfu company about 5.5 kb amplicon
3. redo pTetGFP w taq/ Pfu
4. Miniprep pet26b+sgRNA1 and send for sequencing with primers from positions E1 and E2
5. Save pET26b+USER pcr product in 1.5 ml in misc box. Save 2 best pTetGFP+USER pcr products in 1.5ml in misc box. type up PCR protocol and send (we have to repeat this PCR for the modified pET26b+sgRNA1 now)
6. USER Fuse new pTetGFP+TetR+5varpromoters and transform
7. Get sgRNA in psb1A3 sequenced - use vf vr
8. do co transformation of dcas and sgrna in psb1a3
9. Call NEB about M.Sssi
10. Grow up T9002 in chlor
11. Miniprep T9002 in chlor
12. co transform T9002 in chlor and I751250 in AMP - waiting on t9002 miniprep

19-July

1. MsssI PCRs with new internal primers
2. redo cas9/tale with 7.17 new (F) and (R) and pfu
3. skype with Stef @ 3:00
4. Order seq primers for reporter plasmids
5. run C2,T2,Z2 gel (thermocycler count dracula)
6. mp dcas, dcas-sgrna
7. Design primers for M.SssI from NEB - waiting on dude from NEB on sequence
8. PCR M.SssI from NEB - waiting on primers
9. PCR luxI out of v. fischeri, digest it and ligate it into pDAWN then transform ----- waiting for primers
10. tranform rhl system (c0070, c0071, r0071)

22-July

1. grow NEB mSssI in chlor/kan,kan,chlor, and no AB LB culture tubes
2. Re-transform ZF fusion, pET26b-MsssI ligation
3. Order seq primers for fusion plasmids
4. Set up time to work out bisulfite seq primers with chris asap
5. Redo Cas9 and Tale Round 2 PCR
6. check on pET26b/sgRNA sequencing
7. digest, gel extract digested fragments of pEt26b and sgRNA1
8. grow up pTETGFP for positive control for fluorescence expt
9. grow up pET26b to replenish miniprep stock
10. grow up r0071 (amp)
11. digest T9002 and pSB1K3, gel extract

23-July

1. MPs: first, glycerol stock MsssI, pBAD3, R0071. then miniprep all 3 and pET26b. LIMS. Check ~7:00
2. Miniprep/glycerol stock/send for seq: pBAD reporter plasmid. NOTE: aliquot some off first for fluorescence induction experiment. (dilute back to .05 and induce at 0.1)
3. Miniprep pET26b to replace miniprep in spot D9
4. Check on reporter plasmid re-transformations. Troubleshoot. Re-do transformations as necessary
5. minprep/glycy/make competent NEB M.SssI cells depending on DC's results
6. Troubleshoot gel extraction w Spence. Re-do digestion/gel extraction of pET26b and sgRNA1.
7. ligate, transform pET26b and sgRNA
8. Colony PCR ZF fusion plasmid (1 PET seq primer, 1 fusion primer) and MsssI-pet26b plasmid (1 pet seq primer, 1 MsssI primer) then grow up for miniprep/seq/glycerol stocking
9. Digest verify pBAD miniprep
10. Transform pBAD miniprep into DF's newly competent MsssI strain (use amp + resistance that worked best last night). and test transformation to see that competent cells work ok (psb1a3)
11. Order Primers for COBRA
12. Refine ATC induction protocol with Spencer
13. inoculate M9 cultures with pBAD and with pTetGFP and begin tetracycline/ATC induction experiment. compelling data
14. Watch SAAST demo of SDS Page ~ 1:30pm
15. Transform last night's USER reporter plasmids
16. Digest/Ligate/Transform pET26b with MsssI
17. LIMS minipreps
18. Grow up MsssI in correct antibiotics
19. grow up more T9002 in chlor from glycyrol stock
20. get T9002 from Brad (or re-digest), ligate reporter plasmid, transform

24-July

1. Miniprep ZF fusion plasmid , glycerol stock, send for sequencing
2. Miniprep pET26b alongside
3. Send pBAD3 reporter plasmid for sequencing (4 primers)
4. send minipreps of 44249,27969, and 12612 from positions A2, G5, C10 for complete sequence verification ASAP. These are the exact minipreps used for our fusion plasmids- we ran out of the dCas miniprep, so we need more of that before it can be sentin for sequencing
5. Transform MsssI(+) kan and its NIC - these are in Attila the HUn. Ligations, so use 3 uL and plate all of it undiluted
6. Transform your redo of pET26b+sgRNA1, and NIC. plate all, undiluted
7. grow up more pET26b
8. Continue taking time points of ATC induction (ie 16 hours etc)
9. Redo TALE round 1, then redo TALEround 2
10. More colony PCR of ZF fusion plasmid to try to get more clones
11. Pick Rep Plas colonies for colony PCR/ grow
12. Re-do digestion, gel extraction, ligation of pET26b+sgRNA1 and NIC
13. If you have more sample - re run meth diagnostic in 1.5% TBE Gel (load all!).
14. grow up dcas9 44249 for miniprep
15. test pBAD reporter with arabinose
16. SDS page the zfp
17. order miniprep columns
18. grow up zf 4, 11 in kan. grow up pLci 10 in amp
19. grow up pdawn
20. miniprep T9002 in chlor
21. redo digestion, gel extraction, ligation of T9002 and pSB1K3 and NIC
22. redo transformation of T9002/pSB1K3 and NIC
23. Take reading of sender experiment @ 7

July 25 2013 - July 31 2013

25-July

1. First thing, miniprep glyc stock ZF Fusion plasmid for better yield and ASAP send out for seq again so we can have seq before meeting
2. miniprep dcas9 44249
3. Miniprep/ glycerol stock ZFP4, ZFP11, and pLcI10. send for sequencing (fusion plasmids and rep plasmid)
4. Send pBAD3 reporter plasmid for sequencing (4 primers)
5. Transform Reporter plasmid ligations (pcr tubes in freezer) (6)
6. Transform 3 ZF fusion plasmid into BL21 for protein expression and SDS page, also cotransform with pBAD3
7. PCR purify sgRNA PCR product, digest, gel extract, ligate to pET26b, transform again
8. Redo Pet26b + MsssI Digestion / ligation with new NdeI
9. figures from atc induction
10. Redo dcas round 1, then redo dcas round 2
11. confirm/order BL21 (DE3) cells ~ 7:20
12. Design primers to amplify dCas9 in one round. design first round primers for phusion polymerase.
13. check if other methylation sensitive enyzmes are in target sequence
14. Design primers to add BstUI site before promoter in reporter (site directed mutagenesis)
15. User fuse and transform new TALE with msssi and pet26b
16. Repeat ATC induction experiment @ 1 time point (media, ptet, reporter induced at 0-1280)
17. BstUI assay/verification for pLcI 10
18. try dCas9 in one round with new primers when they come in
19. transform ab's pet26 b msssi ligation and NIC marked L in the fridge
20. Transform Barry Canton's part
21. redo AHL experiment with new AHL

29-July

1. colony PCR on cultures of pet26b+sgrna & NIC using (R)sgRNA1-XhoI from H2 and pet26b(+) Fwd seq from E2 = exp band 414bp
2. glycerol stock ZF fusion 11 in BL21 and dilute and keep culture going to use for protein expression and SDS page
3. colony PCR on cultures 5 reporter plasmids & NIC with primers VF2 and (R) RepPlasSeq1 from i9 = exp band 1650bp
4. Colony PCR on TALE Fusion plasmid with pET26b(+) Fwd seq from E2 and (R)MsssI2 from H8= exp band 3.5kb
5. Glycerol stock / Miniprep the verified cultures/glycerol stock / miniprep I13202 (Canton's sender)
6. PCR user ends onto pet26b+sgRNA1
7. redo colony PCR for tale, pLac, pDNAa (run gel Tuesday morning then grow up successful colonies ASAP)
8. grow up sender/receiver cotransformation

30-July

1. AM: Run Gel on Colony PCRS
2. 2pm: Grow cultures of 5 biobricks, NEB
3. Dilute to .1, then induce with aTc
4. Figure out what went wrong with TetR Sequencing - fix map
5. BEFORE 5PM: Send new reporters, TALE, pet26b+sgrna for sequencing
6. AM : Run Gel on pet26b+sgRNA1 PCR
7. USER fuse and transfrom dCas9+msssi+pet26b+sgRNA, and its (NIC) - ask spencer for cannons
8. Write protocol for protein expression based on spencers
9. Before 5 pm: Acquire from Chow Lab / Cell center all materials for Protein Expression and SDS PAGE
10. minprep pBAD3 (3:30 PM Tuesday)
11. run gels of col pcr of TALE and MsssI, grow up the right clones
12. update lims with seq results; send out type up
13. co-transform zinc finger w pbad3 - commercial bl21. consider doing single transformations on double antibiotic plates as controls
14. transform MsssI ligation and NIC. Transform NEB MsssI - dh5@ max eff
15. try canton sender's media with receiver
16. start cultures in AM
17. Gantt Chart

31-July

1. glycerol stock and miniprep k325259, k325219, k577893, k145279
2. re-do MsssI colony PCR - there were no bands
3. grow up culture of NEB cells
4. look into choosing McrA-, McrB- cells : figure out whats up with NEB's cells
5. Co-transform ZFP11 with pBAD1 in BL21 ~500ng each. Transform just ZFP11 and just pBAD1 in BL21. Transform dcas9 fusions and NIC in DH5@max. Transform NEB Plasmid in DH5@max.

Aug 1 2013 - Aug 7 2013

4-Aug

5-Aug

7-Aug

Aug 8 2013 - Aug 14 2013

8-Aug

9-Aug

12-Aug

13-Aug

14-Aug

Aug 15 2013 - Aug 21 2013

15-Aug

16-Aug

18-Aug

19-Aug

20-Aug

21-Aug

Aug 22 2013 - Aug 28 2013

22-Aug

23-Aug

24-Aug

25-Aug

28-Aug

Aug 29 2013 - Sep 4 2013

1-Sep

2-Sep

3-Sep

4-Sep

Sep 5 2013 - Sep 11 2013

5-Sep

6-Sep

Sep 12 2013 - Sep 18 2013

18-Sep

Sep 19 2013 - Sep 25 2013

19-Sep

25-Sep

Sep 26 2013 - Oct 2 2013

26-Sep

2-Oct

Oct 3 2013 - Oct 9 2013

3-Oct

6-Oct