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