Team:Newcastle/Notebook/LabBookV1/July

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July 1st

  • Description
  • Details of this thing follow
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July 2nd

  • Description
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July 3rd

    Fim Switch
  • Marcia + Lais
    • Digest ran for K1632013 (MP17) and K137058 (MP18)
      37C 1 hour, 64C 20 minutes, 4C rest
      2 copies of each sample digested
      17 + 18 - Arac/FimE
      22 + 21 - FimS/GFP

      For ligation, samples were quantifies on the nanodrop
      MP17 - 264.2 ng/µl
      MP18 - 306.8 ng/µl
      MP22 - 143.9 ng/µl
      MP21 - 117.5 ng/µl


    • Competent cell test kit Calculating cell efficiency
      Ran gel + cut bands

      MP17 - 0.193 g
      MP18 - 0.205 g
      MP21 - 0.202 g
      MP22 - 0.292 g

      Gel purified using the Qiagen purification kit

    • Competent cells testing kit using the iGEM protocol
       Controls
      • DH5α + H20 in LB
      • DH5α + H20 on cm
      • DH5α + GFP on cm
      • DH5α + mRFP on amp
      As well as transforming K1334H

July 4th

    Fim Switch
  • Ansh + Lais
    • Counting cell efficiency
      ---10 50 100
      1 - 2 20 46
      2 - 9 37 73
      3 - 6 36 34


  • Lais, Ansh, Marcia + Sophie
    • Transformation

July 5th

  • Description
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July 6th

    Fim Switch
  • Marcia, Sophie + Ansh
    • Digestion
      MP17
      • 2 µl buffer (cutsmart)
      • 3.8 µl DNA
      • 1 µl Pst1
      • 1 µl Spe1
      • 12.2 µl water

      MP21
      • 2 µl buffer (cutsmart)
      • 8.5 µl DNA
      • 1 µl Pst1
      • 1 µl Xba1
      • 7.5 µl water

      Incubate at 37C 1 hour, 64C 20 minutes, 4C rest

July 7th

    Fim Switch
  • Sophie + Marcia
    • Used E-Gel to purify digest
      20 µl of MP17 and MP21, no dye added
      25 µl water in empty wells
      1kb plus ladder - 10 µl in M lane

      MP17 in lane 4
      MP21 in lane 5

      Digestion wasn't successful, MP21 was smeared in the gel
      Extracted MP17 but not MP21

July 8th

  • Description
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July 9th

  • Description
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July 10th

    Fim Switch
  • Lais, Marcia + Sophie
    • Digestion
      Digested MP18 and MP22

      Ran gel after digest and cut the band
      MP18 - 0.255 g
      MP22 - 0.274 g

      Gel extraction using Qiagen extraction kit

      Ligation
      Ligating at 1:1, 1:3 and 1:5 ratios

      1:1
      • 2 µl buffer x10
      • 1 µl vector (MP18)
      • 1 µl insert (MP22)
      • 1 µl T4 ligase
      • 15 µl water

      1:3
      • 2 µl buffer x10
      • 1 µl vector (MP18)
      • 3 µl insert (MP22)
      • 1 µl T4 ligase
      • 13 µl water

      1:5
      • 2 µl buffer x10
      • 1 µl vector (MP18)
      • 5 µl insert (MP22)
      • 1 µl T4 ligase
      • 11 µl water

      Set overnight (16 h) at 16C

July 11th

    Fim Switch
  • Lais, Marcia + Sophie
    • Transformed cells
      Used Bradley's protocol

July 12th

    Fim Switch
  • Whats this one?
    • 10 ml -> LB final volume
      CAM 2.5 mg/ml -> 12.5 µg/mg

      µg 12.5 x 10 = 2500 x V
      V = 0.05 ml
      = 50 µl

      10 ml -> LB
      50 µl -> Anti B -> CM
      1 colony


      Colony from 1:3 plate transformation started
    Sarcosine Oxidase
  • Bradley + Sophie
      • gBlock Prep
      Followed gBlock preparation protocol
      Centrifuged tube for 3 - 5 seconds
      Resuspended in 50 µl of resuspension buffer
      Incubated for 20 minutes at 50C
      Kept on ice

    • Digestion
      Digested sfGFP plasmid
      • 2.5 µl buffer
      • 2.8 µl DNA
      • 1 µl Xba1
      • 1 µl Spe1
      • 12.7 µl water

      Incubated at 37C 30 minutes, 80C 20 minutes, rest at 4C

      IDT DNA conc on nanodrop was 22 ng/µl

    • HiFi Assembly (Gibson) Master mix, 10 µl
      Linear plasmid, 50 ng, 4 µl
      Resuspended gBlock, 50.2 ng, 2.5 µl
      Water 3.5 µl

      Incubated for 15 minutes at 50C

    • Transformation
      Added 5 µl of the above to competent cells and followed transformation protocol
      Puc19 - ampicillin (NEB 5α cells)
      SOX - chloramphenicol (NEB 5α cells)
      HiFi control - chloramphenicol (NEB 5α cells)
      Antibiotic control - chloramphenicol (DH5α cells)

July 13th

    Fim Switch
  • Marcia + Lais
    • Miniprepped Bradley's stuff, FimE and Lais' promoters
      Following Qiagen protocol
    Sarcosine Oxidase
  • Bradley + Sophie
    • Checking For and Harvesting Colonies
      Transformation plate had colonies
      Most not green, some fluorescent green (due to backbone re-ligation)
      Picked 6 non-green colonies for overnight cultures (using pipette tip)
      5 ml LB in each tube
      2.5 µl of chloramphenicol

July 14th

    Sarcosine Oxidase
  • Bradley
    • Miniprepped overnight cultures

July 15th

  • Description
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July 16th

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July 17th

    Sarcosine Oxidase
  • Sophie
    • Digestion
      Digest of sarcosine oxidase PSB1C3 (from transformed E.coli) for confirmation it's present
      16 µl sample
      2 µl buffer (cutsmart)
      1 µl Xbal
      1 µl Spe1

    • Gel
      Gel run of SOX PSB1C3 digest
      • 3.4 µl 6x loading dye
      • 1 µl Nancy 520
      • 10 µl in each well
      Digestion was good, but 2 lanes showed an empty backbone where the plasmid had re-ligated without GFP or SOX
      Didn't label which digest sample came from which miniprep, so digest and gel repeated to determine the 2 with the empty backbone




      102 and 104 had the empty PSB1C3 backbone

      103 will be used for transformation into BL21-DE3 cells because the band was the strongest, indicating more SOX in that sample

    • Competent cells
      Prepared overnight culture of BL21-DE3 cells from streak plate
      • Picked 'colony' in 5 ml of LB
      • 37C, ~16h

July 18th

    Sarcosine Oxidase
  • Sophie
    • Following competent cell protocol

      Inoculated 40 ml of LB in 250 ml conical flask with 0.4 ml of overnight culture
      Incubated at 37C until OD = ~0.4-0.6


      Time (h) OD
      0 0.042
      1 0.181
      2 0.741

July 19th

  • Description
  • Declan
    • Transformation of the following constructs into DH5a cells:

      • Detector module - Successful
      • Connector 1 (detector cell) - Unsuccessful – Retransform on 20/07/17
      • Connector 1 (processing cell) - Successful
      • Connector 2 (processing cell) - Successful
      • Connector 2 (output cell) - Successful
      • Output module - Successful

      Positive and negative controls as expected
    Sarcosine Oxidase
  • Sophie
    • Transformation
      Transformation of sarcosine oxidase into the BL21-DE3 cells prepared yesterday

      SOX – CAM (used sample 103)
      No DNA – LB
      No DNA – CAM
      SfGFP – CAM

      Overnight 37C, ~16h

July 20th

  • Description
  • Lais
    • Re-transformation of Connector 1 (detector cell) construct
      – Successful
    Sarcosine Oxidase
  • Sophie
    • Transformations were successful

      Overnight cultures prepared
      • 3 x cultures of BL21-DE3 cells
      • 3 x cultures of DH5α cells

      5 ml LB
      2.5 µl of chloramphenicol
      37C ~16h

July 21st

    Cell Free
  • Sophie
    • Harvesting Cells and Prep.
      200 ml overnight culture of cells harvested following Bradley's cell free extract preparation protocol
    Sarcosine Oxidase
  • Sophie
    • Miniprep
      Miniprepped 2 BL21-DE3 and 2 DH5α cultures following the QIAGEN protocol

July 22nd

  • Description
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July 23rd

  • Description
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July 24th

    Cell Free
  • Sophie
    • Continued from 21/07/17
      Followed the remainder of Bradley's cell free protocol from 21/07/17
    Sarcosine Oxidase
  • Sophie
    • Streak Plate Prep
      Prepared streak plate from glycerol stock of DE3-SOX cells - 37C, ~16h

July 25th

    Cell Free
  • Who?
    • Testing cell extract

      Master mix
        1 reaction 5 reactions
      Extract 16 µl 80 µl
      Premix 10 µl 50 µl
      Amino Acids 3 µl 15 µl

      2 with no DNA, 2 with sfGFP (1.7 µg)

      (sfGFP concentration = 185.9 ng/µl, need 1700 ng so 1700/185.9 = 9.1 µl)

    • Reaction
      Master mix 29 µl
      DNA 9.1 µl
      Total volume 50 µl

    • sfGFP
      Master mix 29 µl
      DNA 9.1 µl
      Water 11.9 µl

    • No DNA
      Master mix 29 µl
      Water 21 µl

      Put just under 50 µl in plate wells, put in plate reader
    Sarcosine Oxidase
  • Sophie
    • Streak Plate Prep
      Prepared O/N culture of BL21-DE3 SOX cells and BL21-DE3 cells

July 26th

    Sarcosine Oxidase
  • Sophie
    • What to call this?
      2 ml O/N culture into 200 ml of LB in 1L flask

      DE3-SOX - 2 ml, 200 ml LB, 1 ml CAM (2 flasks)
      DE3 - 2 ml, 200 ml LB

      Incubate for 2 hours

      Added 200 µl IPTG to each flask taking OD readings every half hour

      Time (h) DE3-SOX 1 DE3 DE3-SOX 2
      2 0.681 0.737 0.710
      2.5 0.934 1.065 0.967
      3 1.055 1.273 1.078
      3.5 1.097 1.403 1.126
      5.5 1.242 1.818 1.278

      Started over - prepared DE3-SOX O/N culture again for tomorrow to create a growth curve

July 27th

    Sarcosine Oxidase
  • Sophie
    • 2 ml of O/N culture in 200 ml LB with 100 µl CAM in 2L flask

      Time (h) OD 1 OD 2
      0 0.035 0.034
      0.5 0.048 0.051
      1 0.109 0.113
      1.5 0.256 0.256
      2 0.564 0.554
      2.5 0.845 0.833
      3 0.977 0.962
      3.5 0.990 1.001
      4 1.003 1.012
      4.5 1.062 1.052
      5 1.101 1.095
      5.5 1.144 1.139
      6 1.193 1.188

July 28th

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July 29th

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July 30th

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July

Mo Tu We Th Fr Sa Su
26 27 28 29 30 1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31 1 2 3 4 5 6

What We Got Up To In July

Summary