iGEM 2014 — Team Toronto

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# Team:Toronto/Notebook

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* ## Week 1

  + picked single MG1655 and pTRC99A strains into liquid culture,
    incubated overnight (pTRC99A in LB+Amp)
  - miniprepped pTRC, O/N culture of MG1655
  - MG1655 did not grow on antibiotic plates, so stocks are good!+ prepped DNA for transformation – pCas9 and RFP reporter
  + made MG1655 electrocompetent –
    electroporate and plated transformants, incubated overnight
* ## Week 2

  + transformation of RFP, pCas9, GFP, fmt into NEB5alpha
  - transformants plated and incubated overnight
  - then inoculated into liquid culture, incubated overnight+ overnight culture of MG1655 and pTRC99A
  + Miniprepped RFP and pTRC99A
  - tested restriction enzymes EcoRI, Xbal, SpeI, Pstl
    on miniprep and ran on gel w/ uncut as control
  - cut plasmids were too faint to see – need more
    concentrated DNA in preps!+ O/N cultures of RFP, pCas9, GFP from transformation
  + miniprep of O/N cultures of transformants and pTRC99A* ## Week 3

    + miniprep again, greater volume (3mL)
      for RFP, GFP, Cas9, pTRC99A + nanodrop
    - digest product (w SpeI, EcoRI, cutsmart) and run on gel+ O/N culture from plate of extra biobrick, RFP, MG1655
    + start RFP and MG1655 liquid cultures for the RFP
      spectrum measurements in the afternoon
    - RFP grew slowly and was transformed into NEB5alpha instead of MG1655+ digest 8 samples from yesterday’s miniprep of 8 biobrick cultures,
      load and run gel of digest
    + oligos and gblocks arrived
    + did PCR of pCas9 and pTRC99A chunks
    + Started MG1655 liquid culture for making chemically competent
    - make chemically competent cells, transform RFP into them,
      PCR purification, run on gel* ## Week 4

      + miniprepped pCas9 + pTRC99A (w 7mL of cells)
      + did gel extraction – cut out pCas9 + pTRC digests –
        PCR both as well as nanodrop
      + Mon/Tues = PCR w mastermix,
        Wed = make comp MG1655 and heatshock RFP,
        Thurs/Fri = confirm RFP and Marafini protocol/golden-gate assembly
      - PCR from the Friday before did not work
      - so do lots of minipreps from 10mL cultures and start over, use larger volumes for digests
      DNA eluted into collection tube but saw salt contamination when nanodropped therefore redo miniprep…
    * miniprepped pTRC and pCas9 and then nanodropped –
      results were good so created gel for next day, as well as a digestion
    * digested/ran the gel/extracted, will nanodrop gel extraction – if worked, try PCR again

- ## Week 5

  * Wed. minipreps were good (however possibly from just high free nucleotides), gel extraction failed (no DNA at the end and a lot of salt contamination) – so just PCR from miniprepped plasmids
  * digest and run gel of digest at the same time!
  + PCR of pTRC and pCas9 – have also been digested
  + ran gel! 5 part sample, 1 part loading dye* if PCR worked, digest PCR product w DpnI – got nothing for pCas9 though on the gel (to be re-PCR-ed), but bright bands for pTRC
  * competent MG1655 also done
  * PCR of miniprep C1 #1 of cas9… did 10 x 10uL reaction using gradient temps in 58-68 range
  + run on gel to see if PCR worked
  + PCR gel showed 2 weak bands – either not enough DNA or PCR failed* heat shock transformation for MG1655 w RFP
    plasmid but very few colonies on CM plate- ## Week 6

    * PCR product (???) run on gel, pick most successful PCR
    * DpnI digest following protocol in NEB Gibson manual
      Transform RFP & MG1655, O/N cultures
    * pCas9PCR product visible on gel
    * DpnI digest + PCR cleanup of digest (ended up not having enough DNA)
    * plating of transformed RFP into DH5a
    * processed PCR – ran on gel, DpnI digest also ran on gel, PCR cleanup, nanodrop (worked well)
    * needed to transform pCas9+spacer plasmid & RFP DH5a- ## Week 7/8

      * transform RFP & BL21(DE3)
      * PCR of pCas9, pTRC99A
      * Gibson – pick 8 colonies, make O/N colonies, try to PCR form reaction to get 4-fragment superchunk
      * Maraffini – use Anderson high for 4 fragment
        (phosphorylation + annealing of spacers if we get a lot of DNA), otherwise using Aug 18 miniprep do a BsaI digest
      + got colonies from Gibson* 3 things in parallel:
      + miniprepped plasmids from Gibson transformants from O/N liquid cultures (need to be sent for sequencing)
        , plasmids digested w E+P and to be run on gel
      + making competent MG1655 – transform RFP into it
      + tried BsaI digestion and gel extraction on Stanford-brown pCas9 (for Marafini/Golden-gate),
        does not seem to work even w large volumes* look at plates from transformation, run E+P digests on gel – if the digests look wrong size, do colony PCR on rest of Gibson colonies on plates
      + use fwd/rev primers for cas9 and p!TRC chunks
      + if correct, also PCR the 3-fragment superchunk from the miniprep plasmid by using Cas9 fwd with p!TRC rev primers* try a gel-extraction protocol…- ## Week 9

        * Main thing – **GIBSON ASSEMBLY**
        * screened 8 colonies – miniprepped, EcoRI + PstI digest & wrong product, non-specific assembly
        + extend gblocks (Anderson high + tracr) so more overlap, try Gibson again* did digests of plasmids w one restriction enzyme instead of double digest + run gel (for less confusing results) – used miniprep #1-4
        * made O/N cultures from first Gibson reaction #9-15 – miniprepped and streak out plates
        * minipreps #5-8 from earlier digested w EcoRI and PstO (separately), run on gel
        * take 2 minipreps, do 3 reactions on each – EcoRI only, PstI only, both EcoRI + PstI- ## Week 10

          * miniprep out RFP plasmid to be used for putting biobricks into pSB1C3, will want to screen for non-red colonies at the end of the process
          * unable to do digest because nanoprep of all 3 minipreps was too low
          * primers arrived – ran PCR w new primers
          + also digested RPF
          + run digests on gel (1/2 thickness)* ran the original gblocks w extended gblocks –
            not enough of original gblocks to show up on gel
          * combined PCR reactions of tracr gblocks into one,
            same with Anderson High PCR products
          + purified both
          + PCR purified Cas9 array from a PCR in August
          + purified p!TRC chunk found
          + all 4 to be used for Gibson assembly!* glycerol stocked 3 tubes of colonies from first
            Gibson attempt (#3, 4, 7)
          * transformed Gibson in NEB5alpha, ½ of each reaction
            tube into a total of 4 NEB5alpha – 4 tubes plated
            onto 2 plates each = total 8 plates incubated
          * on Friday, found colonies from the Gibson assembly and
            did colony PCR
          * colony PCR primers were designed to cover the seams
            where the 4 chunks joined
          * mixed the template DNA and biobrick primers for the
            Cas9-array chunk, but did not add the master mix yet
            (done later)- ## Week 11

            * ran Gibson colony PCR on gel – colonies #3 + 6 have correct bands, started O/N cultures of them w cell suspensions from colony PCR
            * miniprep and glycerol stock them (low copy plasmid – do 3uL miniprep)
            * PCR of cas9 chunk – run on gel, see if band is at 4595
            * using RFP miniprep from earlier, digest w EcoRI and PstI
            + linearised plasmid digested same way (if DNA dissolved – was left sitting overnight to dissolve)* nothing on gel at correct size…. only used 1uL of DNA for gel though
            * digest ~1000ng of Gibson miniprep from 2 colonies w EcoRI and PstI, run on gel
            * run 5uL of tube labelled C6\*
            * cas9-array chunk biobrick extension PCR barely worked – not enough DNA after PCR purification
              but tracr chunk biobrick PCR worked
            + digested/ligated into pSB1C3, transformed into competent cells
            + Gibson colonly #3 didn’t assemble correctly, only produced 1 band from E+P digestion* transformed ligation into NEB5alpha – after 2 hour (actual = 10 min) incubation in SOB, pipetted into 2mL LB Cm and grew overnight
            * miniprepped to see if it grew and packed it up for shipping!

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