Team:Penn/Notebook

From 2013.igem.org

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Revision as of 02:53, 27 September 2013

Notebook

  • human practices
    •
  • notebook
    •
    notebook
    • Week 1
    • Week 2
    • Week 3
    • Week 4
    • Week 5
    • Week 6
    • Week 7
    • Week 8
    • Week 9
    • Week 10
    • Week 11
    • Week 12
    • Week 13
    • Week 14
    • Week 15
    • Week 16
    • Week 17
    • Week 18

    WEEK 1

    June 4 2013 - June 11 2013

    Goals:

    This was our first week setting foot in the lab as the Penn iGEM team. We wanted to become more acquainted with the lab and start transforming and growing parts that would be useful in our future project ideas. This week we wanted to brainstorm our project ideas further as well as being more competent in basic molecular biology techniques.


    Accomplishments

    We learned how to make competent cells and performed our first transformations of the summer. We continued to brainstorm potential projects and thought about directions we would like to pursue for the summer. Our idea for a project that would revolve around quorum sensing systems is currently being put into motion. We continued brainstorming for our idea about uses for DNA binding domains like TALE, Cas9, and zinc fingers. We grew up and miniprepped our transformations of various quorum sensing parts and learned how to use a plate reader.

    WEEK 2

    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

    • WEEK 3

    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

    • WEEK 4

    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

    WEEK 5

    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)

    WEEK 6

    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

    • WEEK 7

    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

    • WEEK 8

    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.

    • WEEK 9

    Aug 1 2013 - Aug 7 2013

  • 4-Aug
    1. glycerol stock and miniprep k206500 and Tale18+pBAD1 BL21 cotransformation
    2. send out co trans growth curve
    3. Induction and SDS PAGE on ZF11, TALE 18, and MsssIPET12
    4. -80c lims missing coordinates

  • 5-Aug
    1. Induce M12,T + B1,L4 co trans with .1mM
    2. miniprep MsssiPet12Dh5a, ZF11dh5a, pLci4NEb10, TALE18Dh5a
    3. Grow up Glycerol Stocks of NEB+pBad1, NEB+pLcI4 for induction Experiment
    4. ZF11+pBad1 in DH5@ - use 15 uL
    5. ZF11+pBad1 in T7 express - use 20 uL
    6. ZF11+pLcI4 in DH5@
    7. ZF11+PLCI4 IN T7 express
    8. NEB MsssI + PLcI4 in ER1821 (small tubes in tip box, tape label on top of box - use 50uL)

  • 7-Aug
    1. Look through the miniprep boxes and see which minipreps are relevant to the project and have <10uL left, innoculate fresh cultures of those for miniprepping tomorrow
    2. BsoBI - methylation insensitve isochizmer of avaI
    3. order more cpg methylase
    4. Design gibson dcas9 primers
    5. make alliquots of K, A and C
    6. Make plate LIMS
    7. project abstract
    8. co transform TALE 18 + pbad1 in T7 express, TALE18+pLcI4 in T7 Express, TALE18+pBad1 in DH5a, TALE18+pLcI4 in DH5a, ZF11+pBad1 in DH5a, ZF11+pLcI4 in DH5a
    9. grow up NEB+pbad1 and NEB+pLcI4 for induction experiment
    10. meet with issadore to talk about new device
    11. work on iterating the device

    • WEEK 10

    Aug 8 2013 - Aug 14 2013

  • 8-Aug
    1. Do t7 induction and methylation sensitive digest for zinc finger co-trans
    2. Induce NEBMsssI with reporters for methylation sensitive digest
    3. Prepare presentation for Orkan and Thuy
    4. Do minipreps of: pBad1, NEBMsssI in the fridge
    5. LIMS all the minipreps in random racks in the freezer
    6. pick colonies from yesterday's cotransformations
    7. transform zif 11 + pbad1 in dh5@
    8. transform zif 11 + plci4 in dh5@
    9. transform for methylation repression screen: k584000, k774007, j04450, k079050
    10. design primers to gibson assemble luxi into pdawn
    11. ligate and transform t9002 in psb1ak3

  • 9-Aug
    1. send tale18 for re-do sequencing
    2. induce TALE co trans for digestion assay. choose pLcI4 or Pbad1 and bstui or avaI respectively
    3. transform TALE18, ZF11 in T7 express to grow up tomorrow for SDS Page, glycerol stock, and miniprep
    4. redo zinc finger gel, linearizing,
    5. CUT QUORUM SENSING PROJECT

  • 12-Aug
    1. Miniprep TALE4 , new fusion clone, and send for sequencing asap
    2. Image and send out gel
    3. get mikes compatible GFP vector gorw it up
    4. Send out notes from JMIL
    5. Glycerol stock ZF11 T7, TALE 18 T7 express
    6. Is it possible the co-trans work in BL21 and not in T7 express or dh5a?-- T7express is a BL21 derivative, no reason cotrans should work in one over the other, we're moving it all to one plasmid anyway
    7. make more LB
    8. Miniprep ZF11 T7, TALE 18 T7 express

  • 13-Aug
    1. verify order of (R)MsssIGA2
    2. type up cas9 assembly protocol and go over
    3. send chris biseq primers
    4. Design way to add target to TALE and ZF and MsssI

  • 14-Aug
    1. Send out report on zinc finger linker length, if we can perfectly reproduce it or not.
    2. Learn assembly protocol from JT
    3. Send chow Timeline and what expts, by when, by whom, resource allocation
    4. Incorporate Chow and others suggestions into the powerpoint
    5. need to check TALE binding sequence
    6. SDS page
    7. check which items we need for future protocols
    8. talk to chris about 'perfect primers'
    9. move glycerol stocks in row F of 2012 box to a 2013 box, re-LIMS

    • WEEK 11

    Aug 15 2013 - Aug 21 2013

  • 15-Aug
    1. at 4: grow up addgene dcas9, pET26b+sgRNA lig (1 or 2) for miniprepping
    2. phosphorylate and anneal target oligos for dcas
    3. miniprep and glycerol stock pET26b in T7 (1 and 2), INBNC-mCherry in BL21 (k), intein-mCherry in BL21(amp)
    4. digest pET26b with NotI and XhoI, column purify (3X)
    5. digest pET26b+sgRNA1 with BamHI and XhoI, column purify
    6. ligate target oligos with pET26b/sgRNA backbone (for cas plasmid)

  • 16-Aug
    1. miniprep dcas9, pET26b+sgRNA
    2. phosphorylate/ligate target oligos with pET26b backbone for ZFN/TALE/M.SssI
    3. transform all ligations (TALE/ZFNM.SssI Target, Cas9 Target) in pET-26B
    4. Sort Purchase spreadsheet for Brian
    5. Use Brian's tips to refine the intro slides from the powerpoint
    6. Make detailed plan of when we are spending money, why we are spending it, and how much of it we will be spending
    7. make detailed plan of what figures we need (once plasmids are cloned), what assays to perform

  • 18-Aug
    1. glycerol stock and miniprep backbones with target sequence

  • 19-Aug
    1. PCR sequenced zinc finger fusion, M.SssI, tale fusion (so XbaI and NotI can be used to digest/ligate onto backbone) using primers (F) OnePlasInsert and (R) OnePlasInsert
    2. PCR M.SssI template that DC Gibson assembled with (F) M.SssIGA2 and (R) M.SssIGA3 - see note on JT's dcas protocol about this reverse primer
    3. PCR Cas9 from Addgene plasmid
    4. digest pET26b+target backbone with XbaI and NotI, gel extract
    5. digest pET26b/sgRNA+target backbone with NcoI and NdeI, gel extract
    6. send out new and improved video plan

  • 20-Aug
    1. digest zf, tale, M.SssI PCR products with XbaI and NotI, column purify
    2. order new bisulfite seq primers
    3. work on modularity of one plasmid system
    4. FILM DAY
    5. miniprep dcas and zfn target backbones, elute with water instead of buffer
    6. How else can we clone MsssI into pET26b+Target? (order primers)
    7. ligate pET26b+target (after digestion) with zf, tale PCR products (after digestion); remember the no-insert control
    8. transform pET26b+target + zf, tale ligations into DH5a
    9. Gibson assemble the digested pET26b/sgRNA+target backbone (in-progress B4, conc 12.0) with the PCR'ed cas9 and PCR'ed M.SssI that DC Gibson assembled; call NEB for instructions on how to do this
    10. transform the Gibson assembly into DH5a
    11. repeat assay of backbones+target with AvaI and BglII, using new minipreps, in vitro methylation time course
    12. confirm pet26b+Target lig5 (aka zfn target lig 5) by re-doing the col pcr on both this miniprep and normal pet26b miniprep and running only a small amount on a 2% TBE gel to get distinct bands with 30 bp difference

  • 21-Aug
    1. checking tale and ZF PCR, then digest with XbaI and NotI-HF with cutsmart, ligate to pET26b/target backbone (in-progress box B5, 11 ng/uL) then transform based on typed protocol
    2. gibson assemble and transform CAS plasmid, using backbone pET26b/sgRNA/target (in-progress B4, conc 12.0)
    3. redo miniprep of target+backbones and do methylation test on pet26b+sgran+target. Methylation asay in progress
    4. PCR MsssI, run Gel, Digest with NdeI and NotI, gel extract the larger band
    5. bk is running gel for MsssI PCR
    6. redo Target in Pet26b ligation and transformation
    7. write updated budget for brian
    8. Work on Human Practices Essay

    • WEEK 12

    Aug 22 2013 - Aug 28 2013

  • 22-Aug
    1. check for sequencing on pet26b+sgrna+target so we can go ahead and gibson assemble and transform with cas-msssI
    2. gibson assemble and transform CAS plasmid, using backbone pET26b/sgRNA/target (in-progress B4, conc 12.0)
    3. Do minipreps, digest methylation time course with xbai and avai, do methylation assay with controls
    4. Digest MsssI pcr producrt with ndei and noti, gel extract the larger band
    5. Digest pet26b+target with ndei and noti for msssi ligation, gel extract larger band
    6. make fresh buffer PE
    7. re-do making pet26b+target; look up if overhangs are stable and decide about using backbone digest or re-doing digest and then ligate and transform
    8. Fundraise for next year's team

  • 23-Aug
    1. check for sequencing on pet26b+sgrna+target so we can go ahead and gibson assemble and transform with cas-msssI
    2. gibson assemble (in-progress C7) and transform CAS plasmid into NEB10
    3. Send out methylation time course results
    4. Digest MsssI pcr producrt with ndei and notiHF, gel extract the larger band
    5. bisfulite primers = look at virtual gels, Go over with spencer or mike in AM, send for approval
    6. re-arrange the inprogress box
    7. Digest pet26b+target with ndei and noti for msssi ligation, gel extract larger band
    8. colony PCR on pet26b+target with new (F)Target2Primer and pet26brevseq, include pet26b as control
    9. prepare finalized plasmid maps - PST backbone. cas with his tag from (R)MsssIGA2

  • 24-Aug
    1. colony PCR the Gibson assembled cas plasmid, grow up colonies - no colonies :\
    2. miniprep pet26bsgrnaTarget#3 and tell josh the concentration
    3. ligate MsssI and PST3, transform
    4. miniprep TALE and ZF
    5. Digest TALE, ZF and backbone pet26b+sgRNA1+Target ligation 3 (aka PST3) with NcoIHF and XbaI. Gel extract, ligate, transform

  • 25-Aug
    1. gibson assemble (in-progress C7) and transform CAS plasmid into NEB10
    2. Miniprep Pet26b+sgrna+target 3 (PST3) - borrow column or save culture in fridge for Miniprep tomorrow
    3. digest PST3 with NcoIHF and XbaI, gel extract
    4. Ligate your PST3 with Tale, and with ZF, and NIC, and transform into NEB10
    5. Digest PST3 with NdeI and NcoIHF, column purify
    6. Ligate this PST3 with MsssI in progress box, and NIC, and transform
    7. Order bisulfite primers for ON target
    8. redo outro voice over
    9. prepare "notebook" pages for website

    • WEEK 13

    Aug 29 2013 - Sep 4 2013

  • 1-Sep
    1. COBRA for ON target pilot attempt - methylated and unmethylated
    2. Order BiSeq primers for OFF target
    3. replate T7 transformations of ZF and TALE clones
    4. Transform your overnight ligation mix of MsssI and NIC, plate all on full plates
    5. Prepare ZF and TALE sequencing of all clones for early submission Tuesday AM
    6. Gibson assemble Cas plasmid
    7. Order primers for cloning msssi biobricks
    8. Submit reporter biobricks

  • 2-Sep
    1. Col PCR MsssI and Gibson Assembly
    2. COBRA for ON target pilot attempt - methylated and unmethylated
    3. need to get TaqåI enzyme for COBRA @ target site. waiting response from NEB
    4. write up cobra workflow
    5. get brian to sign travel grant

  • 3-Sep
    1. col pcr gibson assembly
    2. send Cas clone1 for seq

  • 4-Sep
    1. Clone msssi biobrick with your primers
    2. clone msssi in pst3 backbone
    3. methylation time course - GELS FOR PENN APPS
    4. attempt digestion of tale , zf clones
    5. zf target site missing?
    6. do COBRA with taqI enzyme and XhoI enzyme

    • WEEK 14

    Sep 5 2013 - Sep 11 2013

  • 5-Sep
    1. poster presentation 5-630 bodek lounge tues 9/10; meeting with brian next tues 8am?; waiting to hear from orkan and david gdula.
    2. consider alt tale binding sites implications for meth digest
    3. test OFF target cobra primers
    4. call NEB about bisulfite converting plasmid
    5. send fusions for seq
    6. col pcr on biobricks
    7. cas col pcr with proper pos ctrl
    8. essay for bioethics journal
    9. prep interview questions
    10. Mack Institute blurb
    11. travel grant proposal

  • 6-Sep
    1. look at danny's triplicate gels and figure out what s what and redo the graphs
    2. run the gel
    3. call neb and pick up the thing from chris
    4. CAS
    5. use phosphatase on msssi ligation
    6. cobra- digest off target site and get bisulfite kit from chris to see if we can get more complete bisulfite data
    7. order columns/xbaI
    8. wiki
    9. work on magellin
    10. Compare ON primers 3&6 with ON 1&5 for Cobra
    11. PCR OFF target with 3&7 in 50uL for purification and digestion
    12. Digest OFF from 3&7 with TaqI and BamHIHF
    13. presentation for chow

    • WEEK 15

    Sep 12 2013 - Sep 18 2013

  • 18-Sep
    1. write up websites for biobricks, grow up cultures
    2. miniprep biobricks (psb1c3 msssI's and reporters) and go to postoffice
    3. Digest out RFP (wonders f10); gel extract it **alongside gel fragment of the backbone In Progress F1** and ligate to PST BB backbone
    4. Digest MsssI and MsssILinker (in progress box) and ligate to PST BB backbone
    5. Col PCR new ZF target with (F)Target2Primer *note exact primer name
    6. Digest and ligate new ZF target backbone with ZF fusion
    7. Digest and Ligate PST3 with MsssI
    8. Run gradient on biseq primers
    9. Work with MO- who is digesting MsssI and MsssI linker (in progress box) and ligate to PSB1C3 because that colony pcr was sketchy
    10. animation
    11. Essay

    • WEEK 16

    Sep 19 2013 - Sep 25 2013

  • 19-Sep

  • 25-Sep

    • WEEK 17

    Sep 26 2013 - Oct 2 2013

  • 26-Sep

  • 2-Oct

    • WEEK 18

    Oct 3 2013 - Oct 9 2013

  • 3-Oct

  • 6-Oct

    • Retrieved from "http://2013.igem.org/Team:Penn/Notebook"