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