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
Overnight cultures of Ligated products from 09/09/15
