Team:Stanford-Brown/SB16 Notebooks Latex
Latex
Made with Benchling
Project: iGEM 2016
Authors: Taylor Sihavong
Dates: 2016-07-18 to 2016-10-07
Monday, 7/18
ELIAS'S LAB NOTEBOOK
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ELIM orders #234915
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Ordered 9 single stranded oligos
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Primers for linearization of pSB1C3 and fixing Gibson tails
Tuesday, 7/19
ELIAS'S LAB NOTEBOOK
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Fragment #2 came
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PCR fun on BBa_5176017 to amplify pSB1C3
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Took biobrick directly from well 65 plate #6, 2016
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Diluted with 10ul H2O, used 2ul in PCR
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PCR with Q5 high fidelity --> will gel tomorrow
Wednesday, 7/27
ELIAS'S LAB NOTEBOOK
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Gibson overlapping ends: 200nM (A5 hot start, 2xMM)
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P1 & P2: 32bp, Tm = 72˚C
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P2 & P3: 39bp, Tm = 71˚C
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Design for Charlie backbone PCR:
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For primer: 32bp, Tm = 71˚C
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Rev primer: 38bp, Tm = 71˚C
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Cut out backbone from rAIP plasmid:
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5' flanking base: 2079
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3' flanking base: 3725
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Designed with SnapGene at target 55˚C Tm
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Uploaded to "sheet 2" of "melanin" spread on Google Drive
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Will order today
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Design for amplifying Gibsoned latex operon:
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Used SnapGene with target Tm = 60˚C so primers would be long enough to anneal to gibson product & so there would be extra nucleotides outside of prefix and suffix to anneal to
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ELIM order:
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Ordering 4 primers for latex construct & one primer for melanin binding
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Latex: rAIP_latF & rAIP_latR (linearize rAIP backbone for latex operon)
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gib_latF & gib_latR (amplify gib product without adding gibson overlaps)
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gib_ex_latF & gib_ex_latR (amplify gib product with adding gibson overlaps
Friday, 7/29
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mbPCR cleanup done on undiluted Gibson product from Thursday (Taylor)
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DNA reading on Nanodrop was wonky and low concentration (~2ng/ul)
Monday, 8/1
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Ran gel with rAIP linearized backbone for latex operon. Backbone PCRed correctly, but PCR amplification of latex operon post-gibson looked questionable
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Gibson:
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2pt: PCR latex operon + lin rAIP
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8ul of ~12ng/ul
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2ul of ~37.5ng/ul
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4pt: P1/3, P2/3, P3/3, lin rAIP
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3ul rAIP --> 112ng, 86fmol
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1ul each frag --> 100ng, 180fmol
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Plated both gibson products at 1:10 and 1:100 dilutions. Used NEB5alpha cells, will miniprep plasmid if successful
Tuesday, 8/2
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PCR + PCR cleanup done, products sent in for sequencing -- 5 colonies picked
ELIAS'S LAB NOTEBOOK
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Transform:
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Ran colony PCR on 5 colonies from 1:10 dilutions of 2pt gibson on latex operon. Will get sequencing back tomorrow.
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*Made liquid cultures of all 5 colonies, sent out for sequencing --> will miniprep the best one(s) tomorrow based on sequencing data
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Sequencing:
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Premix template ELIM
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1: 4ul and 10ul H2O and 1ul primer
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2: 3ul + 11ul H2O + 1ul primer
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3: 2ul + 12ul H2O + 1ul primer
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4: 2ul + 12ul H2O + 1ul primer
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5: 3ul + 11ul H2O + 1ul primer
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Ten total tubes, one with VF2 and one with VR for each colony
Thursday, 8/11
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DXS Synthase gBlocks arrive
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Hydrated dry DNA with 20 µl D.I. H2O
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Stored in 4°C fridge for later Gibson reaction
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Digested linear pSB1A3 according to iGEM protocol found at:
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http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones
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Corrects ends of pSB1A3 backbone to have the correct length Gibson overlap with the ends of the DXS synthase gBlocks
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Performed Gibson Assembly using NEBuilder master mix to insert two gBlocks comprised of DXS Synthase gene into pSB1A3
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Transformed DH5-α with Gibson product
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Plated 100 µl undiluted onto LB+Amp plate
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Centrifuged transformed cells at 3000rpm for 3 min to pellet, removed 450 µl SOC media supernatant then resuspended cells (2x concentrated )
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plated 100 µl of 2:1 concentrated cells on LB+Amp plate
Sunday, 8/14
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Colony pCR of DXS synthase with 4 colonies
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PCR cleanup nanodrops
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C1: 84.7ng/ul
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C2: 76.2ng/ul
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C3: 76.0ng/ul
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C4: 62.0ng/ul
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8 Sq tubes: F&R for each colony
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Mix: 1ul DNA and 1ul primer and 1.3ul H2O
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Except 1.5ul DNA for CH
Monday, 8/22
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1 plasmid seqeuence confirmed--"Latex Plasmid" (2 rubber transferases). Operon with 3 proteins
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PAGE gel revealed not all proteins were present (proteins being cleaved by latex product)
-->Lyse cells in different way (4 C on plate "Latex Operon").
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Lyse in a different way: sonicate? Ask Jessica
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Sequence verified colonies from "Latex Operon 8/8" (2 and 5) and "DXS Synthase" (2 and 3)
Wednesday, 8/24
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Grow up six liquid cultures overnight at 37˚C in 1.5ml eppendorfs (LB + Chl)
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1 colony/tube for 3 tubes, from cells expressing the latex operon (Gordon's plates)
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1 colony/tube for 3 tubes, from cells expressing DXS synthase (Elias's plates)
Thursday, 8/25
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Grow up 6 liquid cultures overnight for eventual large-scale production in 15ml tubes (6ml LB + Chl, 10ul from each small culture from 8/24)
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Spin down small cultures from 8/24 at 7000rpm for 15 minutes, remove all supernatant and put in -20˚C freezer for protein extraction the next day
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Realisation that DXS enzyme-expressing cells should only grow in LB+Amp and on 8/25 they were grown in LB+Chl
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4 more colonies picked from Elias's DXS plates and put in 1ml LB+Amp overnight to see if the original cells were contamination or are just Chl resistant for whatever reason (?)
Friday, 8/26
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4 DXS colonies grown in LB+Amp had healthy cell growth --> 1ul IPTG added to these 4 liquid cultures plus the 3 latex operon cultures from 8/25
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3 DXS cultures from 8/25 kept in -20˚C freezer just in case we need them later
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Realised that the pelleted cultures were dead anyway so we're just going to do protein extraction.
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6 6ml cultures from 8/25 put at room temp with 6ul IPTG, glucose, and MgSO4; incubating with additives over the weekend
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Protein extraction done on 6 1ml liquid cultures (latex 1, 2, 3, DXS 2, 3, 4 grown up in Amp)
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Soluble fractions collected for all 6 cultures, insoluble fractions collected for DXS 2, 3, 4
Monday, 8/29
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Lumio gel run with 1ul ladder, 5ul lumio buffer/tube, 0.2ul lumio green/tube, 2ul in-gel detection enhancer/tube, 15ul sample/tube
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Run with 3 soluble latex, 3 soluble DXS, 3 insoluble DXS
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Latex bands came out ok, DXS bands all messed up
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For latex, expecting bands at: 24.97kDa (SRPP), 35.79kDa (HRT1), 35.31kDa (HRT2): between rungs 6 and 5 on the ladder
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Ran another gel with only DXS samples: 3 soluble and 3 insoluble, separated by a lane, and then Intern Ryan's sample separated by another lane
Monday, 9/12
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PCR done on pUC19 and DXS synthase with new primers that Trevor! and Amy ordered
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Annealing temp 57˚C for pUC19, ~2600bp
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Annealing temp 54˚C for DXS, ~600bp
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Stored in fridge overnight for cleanup the next morning
Tuesday, 9/13
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PCR cleanup done on pUC19 and DXS synthase: 44.2ng/ul and 33.1ng/ul, respectively
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Gibson done on PCR cleanup products
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1.95ul vector used, 1.78ul insert used
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Gibson results: 1139.5ng/ul
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Colony from old latex operon plates picked and grown in 5ml LB and 5ul Chl overnight
Thursday, 9/15
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Miniprep done of latex operon culture: 76.7ng/ul
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Transformation into T7 cells done with latex operon and DXS plasmids (0.65ul latex operon, 3.5ul DXS)
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Plated on Chl + Kan plates
Tuesday, 9/20
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Thursday transformation failed: realised mistake: puc19 is resistant to Amp, not Kan
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Testing transformation done with T7 cells: latex operon into T7 on Chl plates, and DXS synthase into T7 on Amp plates and Kan plate
Wednesday, 9/21
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As expected, all plates grew except for the DXS on Kan
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Final transformation done with both plasmids into T7 cells (0.65ul latex operon, 3.5ul DXS) on Chl + Amp plates
Thursday, 9/22
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Two total colonies grew successfully in double antibiotic resistance. Both grown up overnight in Liquid Culture (5mL LB +Amp +Chl)
Monday, 9/26
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Small liquid cultures taken to Stanford and grown in 1000ml LB + Amp + Chl
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Culture is split into 3 500ml containers with 333ml of culture
Tuesday, 9/27
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After liquid cultures are grown up, two are taken, centrifuged at 20min, 4˚C, 1200rpm
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The pellets are kept frozen in the fridge as backups for further characterisation and protein purification
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The third liquid culture has 350ul of IPTG, glucose, and MgSO4 added
Friday, 9/30
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The third liquid culture is placed at room temperature to shake and a further round of IPTG, glucose, and MgSO4 is added
Friday, 10/7
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Latex extraction is performed with the protocol from Gordon's lab notebook
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Chloroform and methanol are used to lyse the bacterial cells and precipitate the latex
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A rubbery substance is extracted and put in an eppendorf for later testing
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Rubbery substance is burned as a quick test to see if it is latex
