Team:Penn/Notebook

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                  <a href="https://2013.igem.org/Team:Penn/HumanPractices"><li>human practices</li></a>

<a href="https://2013.igem.org/Team:Penn/Notebook"><li  class="active" style="background-color: rgb(200,200,215);">notebook</li></a>

    <div class="section-title">notebook</div>

 

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         <img src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/APrj2Se2i-/Lynch%20Labs%20PGFI/BE_LynchLabGroup_DSC2811.jpg?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg" class="image"/><b>Goals:<br></b><p>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.</p><br><b>Accomplishments</b><p>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.</p>

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            <li>17-Jun <ol>

            <li>Grow up luxI culture and grow up tetR culture </li>

            <li>Sequence all of the minipreps</li>

            <li>Retransform ptetGFP to see if BL21DE3 cells are competent </li>

            <li>Figure out second control zfp from addgene </li>

            <li>Figure out how to add luxR binding site to target region </li>

            <li>The bisulfite conversion was missing a negative control (bisulfite converted but unmethylated c0051) we'll need this to interperet results</li>

            <li> Miniprep c0078, c0079 + make glycerol stocks check same plasmid with our kit </li>

<li>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)</li>

      <li>19-Jun  <ol>

            <li>  Make competent DH5a && Dam- </li>

            <li>Figure out methylation assays for promoters</li>

            <li>Miniprep psb1A3 && all the 40mL cultures</li>

            <li>Picked many colonies</li>

            <li>Check pTet-gfp under blue light</li>

      </ol></li> </div></div>

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<li>21-Jun<ol>

        <li>troubleshoot plux-luxI pcr</li>

        <li>roubleshoot pdawn-luxI pcr</li>

        <li>made pDawn-tetR pcr work</li>

        <li>miniprep growing cultures, be sure to pick only the glowing ligations </li>

        <li>ransform the correct t9002 amp ligation - determined from gel</li>

        <li>digested t9002 in amp and ptet gfp in amp to identify the correct ligation</li>

        <li>all of the chosen ptet gfp ligations worked, but let's not use 5 || 13 12. troubleshoot t9002 digest</li>

        <li>troubleshoot t9002 digest</li><ol>

            <li>check for contamination of something (run uncut sample, sample + buffer, sample + 1

enzyme, sample + other enzyme, sample + both enzymes)</li>

      <br/>

      <li>24-Jun<ol>

        <li>figure out the primer issues 8</li>

        <li> Pick t9002 colonies for miniprep </li>

      </ol></li></div>

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<ul><li>26-Jun<ol>

        <li>Dam-/dh5a v dam methylation + dpnI && dpnII digest</li>

        <li>Digested/ligated/transformed t9002 in amp </li>

        <li>figure out the primer issues</li>

        <li> Pick t9002 colonies for miniprep</li>

            <li>Troubleshooted and Re-tred PCR for user ends for reporter plasmid</li>

            <li>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).</li>

            <li>Only sequence ptet GFP 11, if verified make sure to note on LIMS that we are only using ptet GFP 11 </li>

                <li>a. Might be strain competition, need to know growth rates</li>

            </ol>

            <li>Re-suspend primers for lux amplifier </li>

            <li>Mini-prep: e0040, psb1a3, r0062</li>    

        </ol></li>

        <br/>

            <li>Think about application of mathylation project in e.coli</li>

            <li>Ceck if plux/GFP-psb1C3 system is working in liquid cultures

                <li>+/- AHL induction at 100nM</li>

                <li>Compare with ptetGFP fluorescence, normal LB fluorescence</li>

                <li>Assay the bvluc and tale1 minipreps in dh5a, dam-, and bl21 cells with msssi and bstuI </li>

                <li>Results: BstUI is blocked by methylation, and cells don’t normally methylate</li>

            </ol></li>

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             <ul><li>3-Jul<ol>

            <li>Streak zinc finger 2 </li>

            <li>Grow up 44251 </li>

            <li>We need to check if the origin of replications are compatible before co transformation</li>

            <li>gel extract promoter fragments from USER PCR</li>

            <li>re-do USER PCR for: TetR, pTetGFP</li>

 

 

            <li>pick up minigenes and primers from the cell center</li>

            <li>Find/make/buy TBE for use in TBE gels (hi-resolution)</li>

            <li>PCR assemble MsssI with USER primers</li>

            <li>get pTetGfp from pcr box and run gel</li>

            <li>PCR purify pTetGFP in eppendorf and LIMs it </li>

            <li>Fab device(s) and begin fiddling with them</li>

            <li>get t9002 seq</li>

            <li>pcr luxI, gel verify, pcr purify, restriction digest, ligate into pDawn (remember NIC), transform</li>

            <li>redo fluorescence experiment using the new cultures as well - report fluorescence per OD.  

 

            <li>Run gel to confirm MsssI PCR assembly/ before meeting-then gel extract the correct band </li>

            <li>redo TetR sequencing of our current miniprep just to be sure</li>

            <li>transform C0179 (lasR without LVA degradation tag) in DH5a, pick colonies, miniprep/glycerol stock</li>

            <li>miniprep T9002/amp from glyc at H10 and C0079 (grow overnight on Thurs to miniprep at same time as C0179)</li>

            <li>Redesign LuxI into pDAWN primers (NdeI, BamHI)</li>

        </ol></li></ul></div>

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         <li>11-July<ol>

        <li>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</li>

        <li>Trouble shoot 1st round DBD pcr's add his tag and flex user site</li>

        <li>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</li>

        <li>miniprep T9002/amp from glyc at H10 and C0079 (grow overnight on Thurs)</li>

        <li>use linearized psb1k3, go ahead and do the digest/ligation t9002 </li>

        <li>redo Step 1 of plan - pcr assembly MsssIwith new primers - pfu. do we have enough msssi to bother with taq?</li>

        <li>do step 7 simultaneously - ie. take some of Part 1 pot into new reaction pot with (7) primers</li>

        <li>Step 5 of plan - PCR pet26b (50ul rxn) (ie. go ahead with zf/tale even though we need to wait up for cas9 backbone)</li>

        <li>streak lawned user fusions of reporter plasmid, grow in SOC for an hour, dilute and use new plates to get single colonies</li>

        <li>trouble shoot gels of step (4) and (3) and (5). figure out which PCR to redo. which to go ahead and digest etc</li>

        <li>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</li>

        <li>image/analyze big TAE gel. Gel extract TALE 2.5kb</li>

        <li>Gel image redo of cas, Tale, sgrna1, and pet26b. </li>

        <li>step 4 - redo sgRNA1 PCR, maybe lower annealing temp?</li>

        <li>Digest 44251 with EcoRI and XbaI</li>

        <li>Gel extract 44251 digest</li>

</br>

<li>15-July<ol>

        <li>3rd try cas9/TALE rd2 and pET26b PCR.</li>

        <li>salvage/Redo MsssI pcr</li>

        <li>redo TetR PCR from biobrick and from Mo's new miniprep</li>

        <li>Write Protocol: Check rep plasmids fluorescence, if there are no NIC colonies: pick 3 colonies of each to grow,</li>

        <li> Rep Plasmids: take dilutions and see if they fluoresce with tetracycine induction, use this to choose clones for glycerol stock, miniprep, and seq. </li>

        <li>test sender receiver in M9 - growing up, need to induce tomorrow (maybe)</li>

        <li>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</li>

      </ol></li>

</br>

      </ol></li>

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         <li>18-July<ol>

        <li>Redo pET26b USER PCR with pfu</li>

        <li>Call taq/Pfu company about 5.5 kb amplicon</li>

        <li>Miniprep pet26b+sgRNA1 and send for sequencing with primers from positions E1 and E2</li>

        <li>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) </li>

        <li>Call NEB about M.Sssi</li>

        <li>Grow up T9002 in chlor</li>

</br>

<li>19-July<ol>

        <li>skype with Stef @ 3:00</li>

</br>

        <li>grow NEB mSssI in chlor/kan,kan,chlor, and no AB LB culture tubes</li>

        <li>Re-transform ZF fusion, pET26b-MsssI ligation</li>

        <li>Order seq primers for fusion plasmids</li>

        <li>Redo Cas9 and Tale Round 2 PCR</li>

        <li> check on pET26b/sgRNA sequencing</li>

        <li>MPs: first, glycerol stock MsssI, pBAD3, R0071. then miniprep all 3 and pET26b. LIMS. Check ~7:00</li>

        <li>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)</li>

        <li>inoculate M9 cultures with pBAD and with pTetGFP and begin tetracycline/ATC induction experiment. compelling data</li>

        <li>Watch SAAST demo of SDS Page ~ 1:30pm</li>

        <li>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</li>

        <li>Transform MsssI(+) kan and its NIC - these are in Attila the HUn. Ligations, so use 3 uL and plate all of it undiluted</li>

        <li>Transform your redo of pET26b+sgRNA1, and NIC. plate all, undiluted </li>

      </ol></li>

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         <li>25-July<ol>

        <li>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</li>

        <li>miniprep dcas9 44249</li>

        <li>Miniprep/ glycerol stock ZFP4, ZFP11, and pLcI10. send for sequencing (fusion plasmids and rep plasmid)</li>

        <li> Transform 3 ZF fusion plasmid into BL21 for protein expression and SDS page, also cotransform with pBAD3 </li>

        <li>PCR purify sgRNA PCR product, digest, gel extract, ligate to pET26b, transform again</li>

        <li>Design primers to amplify dCas9 in one round. design first round primers for phusion polymerase. </li>

        <li>Design primers to add BstUI site before promoter in reporter (site directed mutagenesis)</li>

        <li>Repeat ATC induction experiment @ 1 time point (media, ptet, reporter induced at 0-1280)</li>

        <li>transform ab's pet26 b msssi ligation and NIC marked L in the fridge</li>

        <li>colony PCR on cultures of pet26b+sgrna & NIC using (R)sgRNA1-XhoI from H2 and pet26b(+) Fwd seq from E2 = exp band 414bp</li>

        <li>glycerol stock ZF fusion 11 in BL21 and dilute and keep culture going to use for protein expression and SDS page</li>

        <li>Colony PCR on TALE Fusion plasmid with pET26b(+) Fwd seq from E2 and (R)MsssI2 from H8= exp band 3.5kb</li>

        <li>Glycerol stock / Miniprep the verified cultures/glycerol stock / miniprep I13202 (Canton's sender)</li>

</br>

<li>30-July<ol>

        <li>AM: Run Gel on Colony PCRS</li>

        <li>2pm: Grow cultures of 5 biobricks, NEB</li>

        <li>Dilute to .1, then induce with aTc</li>

        <li> AM : Run Gel on pet26b+sgRNA1 PCR </li>

        <li>run gels of col pcr of TALE and MsssI, grow up the right clones</li>

        <li>co-transform zinc finger w pbad3 - commercial bl21. consider doing single transformations on double antibiotic plates as controls</li>

        <li>transform MsssI ligation and NIC. Transform NEB MsssI - dh5@ max eff</li>

        <li>glycerol stock and miniprep k325259, k325219, k577893, k145279</li>

        <li>look into choosing McrA-, McrB- cells : figure out whats up with NEB's cells</li>

        <li>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. </li>

      </ol></li>

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         <li>4-Aug<ol>

        <li>glycerol stock and miniprep k206500 and Tale18+pBAD1 BL21 cotransformation</li>

        <li>send out co trans growth curve </li>

        <li>Induction and SDS PAGE on ZF11, TALE 18, and MsssIPET12</li>

        <li>Induce M12,T + B1,L4 co trans with .1mM</li>

        <li>ZF11+pBad1 in DH5@ - use 15 uL</li>

        <li>ZF11+pBad1 in T7 express - use 20 uL</li>

        <li>NEB MsssI + PLcI4 in ER1821 (small tubes in tip box, tape label on top of box - use 50uL)</li>

        <li>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</li>

        <li>BsoBI - methylation insensitve isochizmer of avaI</li>

        <li>order more cpg methylase</li>

        <li>Design gibson dcas9 primers</li>

        <li>make alliquots of K, A and C</li>

        <li>project abstract</li>

        <li>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</li>

        <li>grow up NEB+pbad1 and NEB+pLcI4 for induction experiment</li>

        <li>meet with issadore to talk about new device</li>

        <li>work on iterating the device</li>

      </ol></li>

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        <li>8-Aug<ol>

        <li>Do t7 induction and methylation sensitive digest for zinc finger co-trans</li>

        <li>Induce NEBMsssI with reporters for methylation sensitive digest</li>

        <li>Prepare presentation for Orkan and Thuy</li>

        <li>Do minipreps of: pBad1, NEBMsssI in the fridge</li>

        <li>transform for methylation repression screen: k584000, k774007, j04450, k079050</li>

        <li>design primers to gibson assemble luxi into pdawn</li>

        <li>ligate and transform t9002 in psb1ak3</li>

        </ol></li>

</br>

<li>9-Aug<ol>

        <li>send tale18 for re-do sequencing</li>

        <li>induce TALE co trans for digestion assay. choose pLcI4 or Pbad1 and bstui or avaI respectively</li>

        <li>transform TALE18, ZF11 in T7 express to grow up tomorrow for SDS Page, glycerol stock, and miniprep</li>

        <li>Miniprep TALE4 , new fusion clone, and send for sequencing asap</li>

        <li>Send out notes from JMIL</li>

        <li>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 </li>

        <li>verify order of (R)MsssIGA2</li>

        <li>Design way to add target to TALE and ZF and MsssI</li>

        <li>Send out report on zinc finger linker length, if we can perfectly reproduce it or not.</li>

        <li>Send chow Timeline and what expts, by when, by whom, resource allocation</li>

        <li>Incorporate Chow and others suggestions into the powerpoint</li>

        <li>need to check TALE binding sequence</li>

      </ol></li>

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        <li>15-Aug<ol>

        <li>at 4: grow up addgene dcas9, pET26b+sgRNA lig (1 or 2) for miniprepping</li>

        <li>phosphorylate and anneal target oligos for dcas</li>

        <li>miniprep and glycerol stock pET26b in T7 (1 and 2), INBNC-mCherry in BL21 (k), intein-mCherry in BL21(amp)</li>

        <li>phosphorylate/ligate target oligos with pET26b backbone for ZFN/TALE/M.SssI</li>

        <li>Use Brian's tips to refine the intro slides from the powerpoint</li>

        <li>Make detailed plan of when we are spending money, why we are spending it, and how much of it we will be spending</li>

        <li>make detailed plan of what figures we need (once plasmids are cloned), what assays to perform</li>

      </ol></li>

</br>

<li>18-Aug<ol>

        <li>glycerol stock and miniprep backbones with target sequence</li>

</br>

        <li>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</li>

        <li>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</li>

        <li>digest pET26b+target backbone with XbaI and NotI, gel extract</li>

        <li>digest zf, tale, M.SssI PCR products with XbaI and NotI, column purify</li>

        <li> How else can we clone MsssI into pET26b+Target? (order primers)</li>

        <li>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</li>

        <li>transform the Gibson assembly into DH5a</li>

        <li>repeat assay of backbones+target with AvaI and BglII, using new minipreps, in vitro methylation time course</li>

        <li>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</li>

<li>21-Aug<ol>

        <li>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</li>

        <li>gibson assemble and transform CAS plasmid, using backbone pET26b/sgRNA/target (in-progress B4, conc 12.0) </li>

        <li>redo miniprep of target+backbones and do methylation test on pet26b+sgran+target. Methylation asay in progress</li>

      </ol></li>

         <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">