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Revision as of 18:44, 10 September 2015


iGEM Kent 2015


Notebook

June

M T W T F S S
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

July

M T W T F S S
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 2930 31

August

M T W T F S S
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 2627 28 29 30
31

September

M T W T F S S
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

Day 1 Wet lab 22/06/15

  • Made LB plates
  • Made LB broth
  • TIPS autoclaved

    • Day 2 Wet lab 23/06/15

  • Set up Top10 overnight, VS45 overnight, US348 overnight
  • Meeting with advisors to discuss progress

    • Day 3 Wet lab 24/06/15

  • Filter sterilise the buffer
  • 250ml no antibiotic broth, put top10 cells in
  • Incubate until optical density is 0.6
  • Miniprep PSB1C3 plasmid

    • Day 4 Wet lab 25/06/15

  • Transformation
  • Prepare SBC media
  • Digest of PSB1c3 from MS348

    • Day 5 Wet lab 26/06/15

  • Calculated competent cell efficiency
  • Agarose gel formation
  • Gel electrophoresis

    • Day 6 Wet lab 29/06/15

    Transforming linear PSB1C3

    Day 7 Wet lab 30/06/15

  • Miniprep of PSB1A3 plasmid
  • Gel electrophoresis of a large quantity of PSB1C3
  • Meeting with advisors to discuss progress

    • Day 8 Wet lab 01/07/15

  • Gel electrophoresis of the purified DNA extracted
  • Transformation of pSB1A3 circular plasmid to VS45 cells (competent)
  • Digest of all pSB1C3 plasmids

    • Day 9 Wet lab 02/07/15

  • Competent cells transformation efficiency
  • Mini-prep of 1MS340 to get pSB1C3 plasmid
  • Gel extraction of big digest
  • Quantification of the digest. Added sample buffer to each digest

    • Day 10 Wet lab 03/07/15

  • Transformation efficiency

    • Day 11 Wet lab 06/07/15

  • TOP10 cells containing pSB1A3 with limonene synthase were cultured overnight on AMP plates
  • Colonies of VS45 containing pSB1A3 were patched onto chloramphenicol plates
  • Overnight digest of miniprepped pSBIC3 using ECORI and PSTl

    • Day 12 Wet lab 07/07/15

  • Agarose gel of overnight digest - no bands were visible
  • Set up overnight cultures of pSBIC3 and pSBIA3 to be miniprepped the next day
  • Set up an overnight digest of pSBIC3

    • Day 13 Wet lab 08/07/15

  • Agarose gel of overnight digest - no bands were visible
  • Miniprep of pSBIA3 and pSBIC3
  • Overnight digest of pSBIA3 and pSBIC3

    • Day 14 Wet lab 09/07/15

  • Agarose gel of digested pSBIA3 and pSBIC3 - no bands were visible
  • Overnight cultures of MS349 containing pSBIA3 LIMS and MS340 containing pSBIC3 were set up, ready to be miniprepped

    • Day 15 Wet lab 10/07/15

  • Miniprep of plasmids pSBIA3 and pSBIC3

    • Day 16 Wet lab 13/07/15

  • Overnight cultures of MS349 containing pSBIA3 LIMS and MS340 containing pSBIC3 were set up, ready to be miniprepped
  • Overnight digest of miniprepped plasmids pSBIA3 and pSBIC3 using the enzymes ECORI and PSTI

    • Day 17 Wet lab 14/07/15

  • Agarose gel of digested plasmids pSBIA3 and pSBIC3 - it finally worked!!!
  • Miniprep of overnight cultures
  • Gel extraction of both plasmids
  • Analytical gel using Hyperladder I to quantify the plasmid
  • Overnight cultures of Top10 cells containing pVS72

    Day 18 Wet lab 15/07/15

  • Miniprepped pVS72
  • Transformation of VS45 with pVS72
  • Made LB plates with Chloramphenicol and Ampicillin
  • Plated the transformed VS45 cells on the Chloramphenicol/Ampicillin plates
  • Agarose gel of leftover digested pSBIA3 and pSBIC3 from 14/07/15
  • Gel extraction of pSBIA3 and pSBIC3

    • Day 19 Wet lab 16/07/15

  • Counted the overnight colonies of VS45 with pVS72
  • Calculated the transformation efficiency

    • Day 20 17/07/15

  • No wet lab was carried out on this day as we focussed on dry lab tasks

    • Day 21 Wet lab 20/07/15

  • Fresh LB agar plates containing Chloramphenicol and Ampicillin were produced
  • Transformed VS45 with pVS72 colonies were streaked onto fresh Chloramphenicol/Ampicillin plates
    and incubated overnight

    • Day 22 Wet lab 21/07/15

  • Congo red plates with 0.2% L-Arabinose were made
  • 500ml LB broth containing 0.2% L-Arabinose was made
  • Overnight culture of VS45 with pVS72 in LB broth with 0.2% L-Arabinose

    • Day 23 Wet lab 22/07/15

  • VS45 with pVS72 was plated on the Congo Red plates
  • Culture preparation for TEM

    • Day 24 Wet lab 23/07/15

  • TEM imaging

    • Day 25 24/07/15

  • Dry lab day

    • Day 26 27/07/15

  • Transformation of Top10 cells with pVS105 (negative control plasmid), streaked onto Ampicillin LB plates
  • Transformation of VS45 with pVS72, streaked onto Chloramphenicol/Ampicillin LB plates
  • Produced plates containing Congo Red, Ampicillin plates and inducing Chloramphenicol/Ampicillin plates (containing Arabinose)
    and non-inducing Chloramphenicol/Ampicillin plates

    • Day 27 28/07/15

  • Made Chloramphenicol broth and made Ampicillin broth
  • Resuspended Top10 with pVS105 in Amp broth
  • Resuspended VS45 with pVS72 in Chloramphenicol and Ampicillin broth

    • Day 28 29/07/15

  • Miniprepped Top 10 cells containing pVS105
  • Diluted VS45 with pVS72 to OD600 of 0.1 in 3ml of LB
  • Spot plated 5μl of VS45 onto inducing plates (containing Arabinose and IPTG),
    non-inducing plates and Congo red plates (both inducing and non-inducing)

    • Day 29 30/07/15

  • Transformed VS45 competent cells with the negative control plasmid (pVS105)

    • Day 30 31/07/15

  • Weekend cultures of the negative control plasmid in VS45 were set up
  • London meetup

    • Day 31 03/08/15

  • Dry lab

    • Day 32 04/08/15

  • LB plates were made
  • Transformations of cultures were spotted onto Chl + Amp plates (both inducing and non-inducing) and Congo Red.
  • Plates were incubated overnight

    • Day 33 05/08/15

  • Overnight plates were checked for contamination
  • pVS105 and pVS72 were transformed into VS45
  • Transformations were plated and incubated overnight

    • Day 34 06/08/15

  • Overnight cultures were set up

    • Day 35 07/08/15

  • The colonies did not grow overnight, therefore more colonies were set up in the morning, with the OD being checked in the evening
  • The transformation of pVS105 and pVS72 into VS45 was repeated
  • Transformed cells were plated out, and incubated overnight

    • Day 36 10/08/15

  • Plasmids were digested
  • Agarose gel of the digested plasmids was run
  • Colonies were set up as overnight cultures in LB

    • Day 37 11/08/15

  • The OD of the overnight cultures was checked
  • pSBIA3 and pSBIC3 was transformed into Top10 cells
  • Glycerol stock solution of pVS105 and pVS72 was produced
  • Miniprep of pVS72 and pVS105
  • The cultures of both plasmids were spot plated onto separate plates, ready for imaging

    • Day 38 12/08/15

  • pSBIA3 was re-transformed into Top10 cells
  • PCR of pSBIC3 and pVS72

    • Day 39 13/08/15

  • PCR was repeated
  • Scanned the streaked Congo Red plates

    • Day 40 14/08/15

  • Purified the PCR product
  • Digested the PCR product
  • Quantified the plasmid

    • Day 41 17/08/15

  • Ligations of pSBIC3 and pVS72
  • Agarose gel to check the digest worked
  • Ligations were transformed into Top10 cells and plated out
  • Sample preparation for imaging

    • Day 42 18/08/15

  • Overnight cultures of the transformed ligations were made

    • Day 43 19/08/15

  • The overnight cultures were miniprepped
  • Plasmids digested
  • Transformation of plasmids into VS45

    • Day 44 20/08/15

  • Agarose gel of digested plasmids
  • Overnight cultures of ligations of pSBIC3 and pVS72

    • Day 45 21/08/15

  • Overnight cultures were miniprepped
  • Ligations of pSBIC3 and pVS72
  • Gibson assembly of fragments containing CsgAss and Sup35NM
  • Transformation of the fragments into competent cells, incubated over the weekend

    • Day 46 24/08/15

  • Gibson assembly of fragments containing CsgAss, Sup35NM and Cytochrome b562, followed by transformation into competent cells
  • Digest of ligations
  • Agarose gel of digest

    • Day 47 25/08/15

  • PCR
  • Overnight cultures of the Gibson Assembled fragments
  • Digest of Gibson Assembled fragments containing CsgAss and Sup35NM
  • Agarose gel of the digest

    • Day 48 26/08/15

  • Gibson Assembly of fragments containing CsgAss and Sup35NM with pSBIA3 and Gibson Assembly of fragments containing CsgAss,
    Sup35NM and Cytochrome b562, followed by transformation into competent cells
  • Agarose gel of PCR product
  • PCR purification of the product
  • Digest of PCR product

    • Day 49 27/08/15

  • Agarose gel of digest
  • Double digest of plasmids pSBIC3 and pSBIA3

    • Day 50 28/08/15

  • Miniprep of plasmids pSBIC3 and pSBIA3
  • Digest of miniprepped plasmids
  • Agarose gel of miniprepped plasmids

    • Day 51 01/09/15

  • Digest of pVS72 and agarose gel

    • Day 52 02/09/15

  • Miniprep of pSBIC3 and pSBIA3
  • Digest of the miniprepped plasmids
  • Agarose gel was run to check the digest had worked
  • Gel extraction of the digested plasmids

    • Day 53 03/09/15

  • Gibson assembly
  • Transformation of the Gibson assembly products into Top10 cells
  • Digest of pSBIC3
  • Agarose gel of digested plasmid

    • Day 54 04/09/15

  • Overnight cultures of the Gibson Assembled products
  • Gibson assembly of fragments into pSBIA3
  • Transformation into Top10 cells
  • Digested pVS72
  • Agarose gel
  • London meetup
  • Stacey Symposium

    • Day 55 05/09/15

  • Gel extraction
  • Digest of plasmids and Gibson assembled products
  • Agarose gel of digests
  • University of Kent open day
  • Presentation at London iGEM meetup

    • Day 56 07/09/15

  • Transformation of pSBIC3 and pSBIA3 into Top10 cells from the iGEM distribution kit
  • PCR
  • Transformation of ordered plasmids into Top10 and VS45
  • Miniprep of pSBIA3
  • Digest of pSBIC3 and pSBIA3, followed by an agarose gel
  • Diagnostic digest of pVS72 and pVS72 with cytochrome b562, followed by an agarose gel

    • Day 57 08/09/15

  • PCR purification
  • Ligation
  • Gibson Assembly
  • Transformation
  • Made plates

    • Day 58 09/09/15

  • Miniprep of pSBIC3
  • Digest of pSBIC3 and heat inactivation of the enzymes at 80ºC
  • Ligation of pSBIC3 with PCR products
  • Overnight cultures of pSBIC3 in Top10 cells

    Day 59 10/09/15

  • Miniprep of pSBIC3
  • Digest of plasmid and heat inactivation of the enzymes used
  • Ligation of pSBIC3 with PCR products
  • Gibson assembly of pSBIC3 with ordered fragments
  • Competent Top10 cell production