Team:Toronto/Notebook

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!