Team:ETH Zurich/Notebook/Text

22/06/2015

  • Interlab study:ligation and transformation
  • Redo transformation that failed
  • Order primers, Antibodies and lactate kits

23/06/2015

  • Send trafos (bricks and Interlab parts) for sequencing
  • FACS training
  • Colony PRC for trafos
  • Make cryostocks of interlab positive and negative control
  • Design and order lldRO versions (incl lacO)
  • transform again InterLab ligated constructs (twice each)

24/06/2015

  • Colony PCR for InterLab constructs
  • Send InterLab constructs for sequencing
  • Annexin V part : put parts together with backbone
  • Amplify lldR from the genome
  • Make overnight culture for GFP cells
  • Make cryostocks of the 3 constructs (3 replicates)
  • Retransform InterLab control I20270?
  • Design and order all remaining constructs/primers

25/06/2015

  • Got labelled annexin
  • ON culture for I35104 and the three InterLab promoters (15mL in total) from picked colony
  • Run lldR PCR on gel and purify the construct!
  • Look into sTRAIL handling (prepare for use)
  • GFP competent cells

26/06/2015

  • Miniprep Of InterLab study
  • Digestion of InterLab-purification
  • lldP amplification from the genome--> didn't work!
  • Amplify INP from the biobrick and purify from gel
  • Alliquoting of TRAIL

29/06/2015

  • Throw away the incorrect parts
  • Transform TOP10 and gfp strains for efficiency study
  • Measure concentration of fG0008-fG0012
  • Ligate InterLab fragments
  • InterLab transformation
  • Get Omp-8 cells
  • TOP10 ON culture

30/06/2015

  • Make TOP10 cryostock
  • Transformation efficiency study
  • Colony PCR Interlab
  • Interlab sent to sequencing
  • Measure conc of lldP
  • Transform pSEVA
  • Make new LB+CM plates
  • Make an ON culture from c13


01/07/2015

  • Passage cells
  • Pick up Omp-8
  • Use B0032 (sequenced) for negative control of InterLab
  • Make new LB
  • Colony PCR
  • Send device to sequencing
  • Omp-8 ON culture at 25ºC
  • pSEVA ON culture
  • Competent cells

02/07/2015

  • Miniprep pSEVA
  • Send one sample of pSEVA for sequencing
  • Repeat interlab 2 colony PCR (all negative..)
  • Depending on sequencing results: throw out or correctly label the cryo tube which is now called p32-1
  • Sample resubmitted for sequencing with reverse primer oiG002
  • Remake Omp-8 preculture
  • Make TBF buffer

03/07/2015

  • Mammalian cell passaging
  • Order oligos
  • Amplification of annexinV
  • Digestion of pSEVA low copy
  • TPR digestion: failed partly
  • Digest of InterLab 2

06/07/2015

  • Digest pSEVA371
  • SOC medium
  • Gibson assembly annexin
  • Plan mammalian experiments with TRAIL
  • Transform interlab, annexinV-pSEVA, pSEVA371, pSEVA271
  • Overnight culture of Omp-8 at 16°, 25°, 37°
  • Digest pSEVA371

07/07/2015

  • Passage cells
  • Asked Tania
  • Comment with Margaux the mammalian experiments
  • Cryostocks
  • Primer design
  • Colombia -> protocol sent
  • PCR for lldP-lldR

08/07/2015

  • Make LB-agar and LB-medium
  • pSEVA miniprep
  • pSEVA digest and purification with miniprep
  • Purify lldR-lldP fragments (fridge) (digest the template first)
  • PCR for remaining INP-Annexin fragments
  • 2% agarose gel for remaining INP-Annexin fragments
  • Make ON cultures of all useful BioBrick transformants for cryo stocks

09/07/2015

  • Start mammalian experiments+ TRAIL
  • Shopping list
  • Colony PCR annexinV colony
  • Competent Omp-8
  • Mammalian cell FACS
  • Gibson assembly
  • Transformation INP-An, lldP-lldR, AnV, p27 and p30
  • Make LB
  • Preparation of interlab study

10/07/2015

  • Colony PCR
  • Cryostocks (still missing 27 and 30)
  • InterLab FACS done!
  • Sequencing
  • Make ITA buffer

13/07/2015

  • Compare results: no results
  • Colony PCR of lldR-lldP
  • GA for lldR-lldP diluted
  • pSEVA271 digestion and miniprep
  • Preparation of lldR-lldP plates
  • After meeting planning of experiments
  • ON culture of AnV and INP-AnV

14/07/2015

  • Cryostock of interlab device 2
  • lldR-lldP colony PCR
  • Miniprep of ON of AnV and INP-AnV, send to sequence
  • TPR re-do
  • BioBrick assembly of luxI-term
  • Gel-purify all PCR products from monday
  • Evening: set up mammalian cells with christian
  • pSEVA ON for cryostock
  • BB assembly ligations overnight

15/07/2015

  • Start TRAIL and E Coli mammalian experiments, monitor all day
  • Colony PCR for new colonies of AnV
  • ON cultures of AnV
  • lldR-lldP miniprep
  • Redo digest for luxI-term assembly (C0161)
  • Transformation of bb-assembled stuff
  • Send for sequencing: the "lldR-lldP" plasmid (once with oiG0003 and once with oiG0004)
  • Make overnight culture from cryostock of INP-Annexin_2
  • ON of GFP cells for co-culture
  • Gibson Assemble Plasmids p62-81
  • Transformation of gibson assembled plasmids

16/07/2015

  • Make cultures of BB assembled, colony PCR
  • Make cultures of p62-81, colony PCR, make overnight cultures
  • Pick up sequencing labes from shop- afternoon (let grow for a long time): miniprep a lot of INP-Annexin, send for sequencing with oiG0004
  • Omp-8 transformation efficiency
  • Set up mammalian cells and bacteria for lactate experiment
  • Mini-prep C0161 and B0012
  • Digest C0161 and B0012
  • Overnight culture for interlab

17/07/2015

  • Set up reader plate
  • Gibson assembly for lldR w/o lldP
  • Transform and plate lldR constructs
  • Lactate experiment: change medium at 12:30
  • Make cryostocks of all things to sequence (annexin and all assemblies)
  • Send all things for sequencing
  • Use TOP10 culture for lactate experiment
  • Repeat co-culture 14:00-16:00
  • Redo digest of C0161 and B0012 (includde test of enzymes)

19/07/2015

  • Set up overnight cultures of cryos made on friday plus maybe more of each assembly from 0715 plus from assembly on 0717


20/07/2015

  • Check sequencing results
  • Data process: lactate
  • Redo digest to test enzymes
  • Send stuff to sequence
  • New fragments with new fragments
  • Make colony PCR
  • Throw away stuff from cryostocks
  • Make overnight cultures of what is missing: (p72 and p73)
  • Plan mammalian experiments to know how many cells we need each day
  • Process data (FACS, InterLab and lactate)
  • Make o/n cultures for interlab in triplicates

21/07/2015

  • Early do interlab and InterLab sumitted
  • Keep making new fragments with new primers
  • If sequencing is correct, make fragments for PL(5)
  • Send p72 to sequence
  • New fragments: DPN1 digest, gel, purify from gel
  • Miniprep stuff
  • Continue TPR, ligate and transform

22/07/2015

  • TRAIL experiment start at 9:00 fro 3T3
  • Repeat transformation of lldR and TPR
  • Miniprep c44 and c45 and INP-Ann and p72 to have more plasmid
  • Sample organisation study
  • Colony PCR and make ON culture of lldR andTRP(12_114_34)
  • Send 50 for sequencing with different primer
  • Miniprep p73 variants for sequencing
  • Colony PCR
  • TRAIL experiment 15:30-17:00 FACS machine
  • Continue TPR assembly
  • Digest BioBrick backbone
  • Annexin buffer binding made new
  • TOP10 ON

23/07/2015

  • Colony PCR of GFP-INP-Annexin
  • Colony PCR of lldR, 12_117, TPR
  • Make more LB
  • Digestion Annexin- bb--> no band on annexin
  • Send TPR for sequencing
  • ON culture of AnnexinV and B0012
  • Making Competent Top10 cells

24/07/2015

  • Miniprep annexin
  • Digest INP-An, B0012
  • Retransform TPR minipreped yesterday
  • Amplify TRP to use in assemblies if sequencing is correct
  • Send a lot of things for sequencing (lldR, 12_117)
  • Do gibson assembly for pl5, transform

26/07/2015

  • Precultures

27/07/2015

  • Experiment with Jurkat cells started (lactate)
  • Ligation AnV-BB
  • Ordered new lldR-lldP plasmid gblocks
  • Miniprep ON cultures (TPR and PL(5))
  • Send ON cultures (TPR and PL(5))for sequencing

28/07/2015

  • Make more TAE
  • Miniprep lldR ON cultures
  • Send lldR for sequencing
  • lldR gibson assembly
  • lldR transformation
  • ON cultures of AnV(c73), INP-AnV(c55), PL(5)(cryos 2b, 1, 2a), PL(7), TOP10 cells with no plasmid (c13)

29/07/2015

  • Book plate reader
  • Think about PS-coated
  • ON Cultures annexin in BBBB
  • Experiment Annexin-INP in the membrane
  • Order one TPR in IDT

30/07/2015

  • Digest of INP-Annexin (failed)
  • Digest of luxI and B0012 (failed)
  • ON cultures pSEVA 271 and 371
  • ON cultures of B0012 (c27), INP-AnV, luxI
  • AnV in BioBrick BackBone sent to sequencing
  • Western blot for annexin-inp
  • Make ON cultures from p101 (lldR-lldP) plates
  • Make new LB and LB-agar
  • ON cultures of PL(6)
  • Mammalian set up

31/07/2015

  • Miniprep pSEVA
  • Miniprep PL6
  • Miniprep and digest of B0012 (c27), INP-AnV and luxI and ligate
  • TRAIL jurkat and 3t3
  • Lactate all lines + calibration curve
  • PCR for fragments 74-80, 97, 98
  • Digest test

01/08/2015

  • Gibson assembly lldP-lldR
  • Transformation

02/08/2015

  • Colony PCR of up to 20 colonies with primers oi3 and oi19
  • Make ON cultures, two of every positive colony from the PCR

03/08/2015

  • Order gblock
  • Track selection
  • Cryostocks and miniprep lldR-lldP
  • Sent stuff for sequencing
  • Lactate evaluation (FAIL)
  • Try PCR (p24 + o61/o62 = f64) with Q5 2x Master mix (783bp fragment) at 62, 64 and 66°C (FAIL)
  • CRYO STOCKS!!!! miniprep and send for sequencing: lldR-lldP
  • 4pm meeting doctor
  • Order primers

04/08/2015

  • Start cultures of lldR and lldR-lldP for electrocompetent cells (let grow to OD 0.6)
  • Prepare everything for lactate based experiments
  • cultures of backbone 371
  • order cryotubes
  • transform pl3 into intermediate plasmid strain
  • make gibson assembly again and transform

05/08/2015

  • Crystock of pSEVA
  • Miniprep pSEVA+ failed digest
  • Colony PCR of PL3
  • GA for lldP-lldR (old-old one-with low concentration-): one 1h 45ºC, the other 2h 50ºC
  • Seeding new mammalian cell line
  • ON culture of pSEVA
  • Redo Gibson Assembly of p64 and p73, transform and incubate o/n

07/08/2015

  • Miniprep p50
  • Assemble lldPR, PL3, PL4, PL5, INP-an and transformation
  • Change concentrations and repeat the experiment of the transformants with PL3 and lldR

08/08/2015

  • Colony PCR of all Trafos from Friday
  • Redo assemblies of INP-AnV and PL(5)
  • ON of INP-AnV and PL(5) from assemblies from friday
  • Transformation of assemblies

09/08/2015

  • Redo assemblies of PL(3) and PL(4)
  • Trafo of assemblies
  • Miniprep ON from Saturday
  • Make ON of c89-c98

10/08/2015

  • Colony PCR of PL(3) and PL(4)
  • Make ON cultures of PL(3) and PL(4)
  • Send things for sequencing (INP-AnV and PL(5))
  • Assemble PL(9)
  • Transform assembly of PL(9)
  • Lactate calibration curve
  • Lactate experiment mammalian cells
  • Data evaluation
  • Overnight cultures of INP-annexins and c89-c98
  • Lactate experiment e coli

11/08/2015

  • Cryostocks PL3, Pl4, lldr, miniprep, sent for sequencing
  • Colony PCR PL9
  • Lactate experiment with bacteria twice
  • ON of pseva271
  • Colony PCR of PL(9), make ON if it worked (didn't work)
  • Throw away not confirmed cryos
  • Redo PL9 assembly and trafo
  • Do new assemblies with just arrived primers

12/08/2015

  • Miniprep and digest bb
  • PL4 and PL3-3 sent for sequencing
  • Colony PCR of PL(9), didn't work
  • Redo assembly for PL(9) with different backbones and transform
  • Assemble and transform lldR-lldP, annexinseva, annexinbb, inpyfp
  • TRAIL experiment
  • Lactate addition experiment with bacteria twice


13/08/2015

  • Colony PCR
  • Replan mammalian cell experiment: why TRAIL is not working?

14/08/2015

  • Cryostocks of new cultures
  • Colony PCR of PL9
  • Miniprep of lldrp, pl9, pl3, pl4 and AnnBBBB and sent some for sequencing
  • Miniprep of lldrp, pl9, pl3, pl4 and AnnBBBB and sent some for sequencing
  • Preparation of apoptotic cells (TRAIL)
  • Co-culture HL-60 and bacteria
  • Split cells

15/08/2015

  • Lactate dose-response experiment with bacteria
  • Darmstadt visit

16/08/2015

  • Lactate dose-response experiment with bacteria
  • Darmstadt visit

17/08/2015

  • Send for sequencing lldrp, pl9 etc
  • Good ones PL3 measured at high conc
  • Bad ones (65-68) PL(3) measured at same conc
  • Microscopy TRAIL
  • Miniprep
  • Repeat lactate measurement for cells at 6h
  • Make overnight cultures of PL(3) versions, top10, lldr, lldplldr

18/08/2015

  • Bad ones of PL(3) in plate reader at 25mM, 6.3, 3.1, 390uM, 100uM, 10uM
  • Make fragments from PL(9)
  • Measure lactate from mammalian cells from yesterday (morning before other exp.)
  • Make competent cells
  • Gp62, p69, p70 and p71 again with high concentration of lactate at gain 70
  • More minipreps

19/08/2015

  • p68, p66, p67 and p63 for all concentrations of lactate
  • Send things for sequencing
  • Remake fragment f48 failed

20/08/2015

  • Make SOC medium
  • ON cultures pSEVA and annexinV, PL5, INP AnV and AnnexinV in BBBB
  • Transform PL4 in lacI
  • Remake fragment f48 and reassemble/retransform PL(4)a
  • Maybe redo good plasmids two per plate whole range (all)
  • Double transform PL(4) into lacI strain (c29)
  • Double trafo if sequencing is good (PL(4) and PL(3) versions into lldPlldR)

21/08/2015

  • Colony PCR of double transformants and PL4
  • Miniprep pSEVA271 and digest (133ng/uL)
  • Western blot to check annexin presence
  • Buy enzymes for GA

22/08/2015

  • GA buffer
  • Gels for PCR
  • Cryostocks of double transformants
  • Split mammalian cells
  • Picture of western blot
  • Fragments lldR-lldP--> didn't work. Plan again.
  • Miniprep PL4--> not enough concentration for sequencing. Wait until new kit arrives

23/08/2015

  • Western blot for AnnexinV in BBBB and pSEVA
  • Lactate experiment with p75 and p77


24/08/2015

  • Take lldpr operon from genome with oiG12 and oiG19. use phusion PCR with 5 min heating in the beginning from a resuspended colony or from cryotube, best is empty TOP10.
  • Western blot with higher concentration of protein.
  • Split cells

25/08/2015

  • Get beads with streptavidin
  • Colony PCR lldP-lldR (oiG76 and oiG75) and make cultures
  • Check 3T3 cells and split
  • Digest B0012 for obtaining BBBB
  • INP-AnV, pl5 fragment
  • Experiment with beads (microscopy and FACS): we have red fluorescence in the beads!
  • Make LB

26/08/2015

  • p69 with and without lactate experiment
  • miniprep INP-Annexin, PL5, lldRP, PL4a colonies
  • Send lldRP to sequence and test digest
  • Digest, ligate and transform INP-Annexin and PL5
  • Primer design
  • Make agar

27/08/2015

  • p62 and p70 whole range with and without lldR
  • Split mammalian cells
  • Miniprep more lldPR
  • Colony PCR INP-Annexin and PL5 in BBBB
  • Discuss presentation
  • Make fragments for restriction plan for lldPR
  • Purify from gel: amplifiaction of gblocks. fail.
  • Overnight cultures
  • Found HER2 cells
  • Got superkillerTRAIL
  • Double transformation of LP3 and PL4 versions into lldPR if sequencing is correct

28/08/2015

  • Beads and antibody experiment for everything related to annexin (incl from BBBB)
  • Miniprep p68
  • Colony PCRs for double trafos
  • TRAIL 2h and 4h with Jurkat and HL60. use bacteria to detect
  • Add PL(5) cells to mammalian cells after TRAIL
  • Purify fragemtn for restriction plan
  • Digest and ligate lldPR restriction plan
  • Transform lldPR restriction plan
  • Test PL9 response to AHL
  • Test p71+-lldR for whole conc range
  • Mini prep and send for sequencing: PL5 and INP-Annexin in BBBB

29/08/2015

  • Make fragments for GA
  • Colony PCR reeated for p71+lldPR
  • Colony PCR for lldPR restriction plan
  • Make cryos of all double trafos with lldPR
  • Make overnight cultures of all double trafos with lldPR and do GA with them
  • Set up first presentation draft
  • Set up PCR for PL12 fragment
  • Digest more backbone


30/08/2015

  • Got the cryos for Stockholm
  • Digest more backbone
  • Colony PCR lldRPres, PL1, PL10

31/08/2015

  • Pick up stuff from shop
  • lldP-lldR with restriction plan, PL1 and PL10 sent to sequence
  • Digestion of backbone
  • Colony PCR --> nothing is working again
  • GA of PL10 and PL1
  • Lactate experiment
  • Pick up and thaw SKBR-3 cells

01/09/2015

  • Colony PCR of p71
  • Fragment purification for GA
  • Mammalian lactate experiment
  • Beads experiment: failed
  • Take a look at lldP-lldR under the microscope (different shape)
  • GA of PL10

02/09/2015

  • Colony PCR of PL10--> nothing works (desperation)
  • Lactate experiment with bacteria
  • Measure lactate in mammalian cells: collect up to 24h
  • Make overnight cultures

03/09/2015

  • Colony PCR of PL12, and all versions of lldRPres and PL3 and PL4.
  • Do the presentation for EPFL
  • Human practices collaboration with EPFL
  • Prepare fragments for PL2
  • Analyze data for mammalian lactate experiment
  • Prepare more samples for characterization of our promoters.

04/09/2015

  • Purify fragments
  • Split cells
  • Set up new characterization experiment for lldRPres
  • ON cultures for lldRP experiment

05/09/2015

  • Chip experiment
  • Experiment to see green fluorescence in minimal medium
  • Amplification and purification of fragments for BioBrick
  • Lactate experiment
  • ON cultures of bacteria sent by Stockhoml, lacI, p76, p74, p73 and TOP10.

06/09/2015

  • Assembly and transformation of promoters in biobrick
  • Lactate experiment
  • Transform GFP into Stockholm cells and fragments into BBBB
  • Split cells
  • ON cultures of PL5 in BBBB, PL12, TOP10
  • Colony PCR

07/09/2015

  • Miniprep and send for sequencing: PL2, PL12, PL10
  • ON cultures of PL12, PL5, RFP, PL10, TOP10
  • Repeat fragments

08/09/2015

  • Beads experiment with PS and Ab
  • Competent cells
  • Lactate experiment for Jurkat and SK-BR-3
  • Traansform Stockholm cells with GFP again

09/09/2015

  • Colony PCR of things in BioBrick BackBone
  • Do new assemblies in BioBrick, previous didn't work
  • ON cultures

10/09/2015

  • Preparation of fragments, GA of PL2 and transformation
  • Colony PCR
  • Digest of BioBrick BackBone.
  • Ligation and transformation of parts for Registry (again)
  • ON cultures
  • Preparation of mammalian cells

11/09/2015

  • Mammalian cells experiment
  • Colony PCR of BBBB. Didn't seem to work...
  • TRAIL experiment for concentration range + FACS
  • Incorporate bacteria in apoptotic mammalian cells + FACS + microscopy
  • Backbone cultures
  • Introduce lldR and lldRP into bacteria
  • Do Stockholm experiment
  • ON cultures

12/09/2015

  • Miniprep BB BB
  • ON cultures
  • Digest BB, digest material for biobrick
  • Transform bacteria

13/09/2015

  • Colony PCR of BBBB: some positives!
  • Co-culture Jurkat and bacteria
  • Induction of apoptosis and look in the microscope the result, with three different concentrations of Annexin Binding Buffer and in three temperatures.
  • Prepare Stockholm cells
  • Lactate measurements

14/09/2015

  • Make fragments with newly arrived primers
  • Colony PCR of biobricks: we have all the promoters now
  • Stockholm experiment with mammalian cells
  • Miniprep BBBB

15/09/2015

  • TRAIL experiment: measure lactate concentration in apoptotic cells and test again ANnexin binding. --> no success
  • Colony PCR of the backbone.
  • ON cultures of lldP, lldPR in BB
  • Co-culture HL-60 and bacteria

16/09/2015

  • Colony PCR
  • ON culture of pSEVA371, for 100mL
  • Lactate measurement
  • Last try for assembly of PL14 and PL1

17/09/2015

  • Be sleep deprived
  • Cry and panick
  • Trying desperate measures to get the AND gate
  • Many plate reader measurements
  • Colony PCR
  • ON cultures

18/09/2015 17/09/2015

  • Becoming insane
  • Data analysis
  • Plate reader still running