Team:ETH Zurich/Notebook/Text

06/22/2015

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

06/23/2015

• 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)

06/24/2015

• 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

06/25/2015

• 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

06/26/2015

• 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

06/29/2015

• 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

06/30/2015

• 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

07/01/2015

• 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

07/02/2015

• 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

07/03/2015

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

07/06/2015

• 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

07/08/2015

• 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

07/09/2015

• 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

07/10/2015

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

07/13/2015

• 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

07/14/2015

• 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

07/15/2015

• 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

07/16/2015

• 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

07/17/2015

• 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)

07/19/2015

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

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07/20/2015

• 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

07/21/2015

• 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

07/22/2015

• 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

07/23/2015

• 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