iGEM Kent 2015

Notebook

Day 1 Wet lab 22/06/15

On our first day in the lab, we began by autoclaving equipment ready for use throughout our project. Specifically, we made LB plates, LB broth and autoclaved pipette tips

Day 2 Wet lab 23/06/15

We set up overnight cultures of Top10, VS45 and MS348 cells. We then met with our supervisors to discuss our progress

Day 3 Wet lab 24/06/15

First, we made and filter sterilised MES buffer. Then we added our Top10 cells to 250ml of LB broth containing no antibiotics. The culture was then incubated until the OD₆₀₀ was 0.6. Finally, we miniprepped pSBIC3 out of MS348

Day 4 Wet lab 25/06/15

We set up an overnight digest of pSBIC3 ready to be run on an agarose gel in order to check that we had obtained the correct plasmid from the cells. We then transformed pSBIC3 into our competent Top10 cells. Next, we plated the transformations out onto Chloramphenicol plates and incubated them at 37ºC overnight.

Day 5 Wet lab 26/06/15

We checked the plates from overnight and calculated the competent cell efficiency. We then ran an agarose gel of the overnight digest. We decided on the layout for the wiki.

Day 6 Wet lab 29/06/15

On this day, we transformed linear pSB1C3 into our Top10 cells. We also set up overnight liquid cultures of MS349 cells containing pSB1A3. We set up a large quantity digest of pSBIC3, ready for gel extraction the following day.

Day 7 Wet lab 30/06/15

We miniprepped pSBIA3 and set up an overnight digest of it. We then ran the overnight digest of a large quantity of pSBIC3 on a gel. Finally, we had a meeting with our supervisors to discuss our progress.

Day 8 Wet lab 01/07/15

We ran a gel of purified pSBIC3 ready to be extracted, we then set up a digest of our entire stock of pSBIC3. We also transformed pSB1A3 into competent VS45 cells and plated it onto mixed Chloramphenicol and Ampicillin plates. We set up overnight cultures of MS340 containing pSBIC3.

Day 9 Wet lab 02/07/15

On this day we miniprepped pSBIC3 out of MS340. We then ran an agarose gel of the "big digest" and carried out a gel extraction procedure. We then quantified the digested material.

Day 10 Wet lab 03/07/15

We calculated the transformation efficiency of pSBIA3 in VS45 cells.

Day 11 Wet lab 06/07/15

We set up overnight cultures of Top10 cells containing pSB1A3 with limonene synthase on AMP plates, and colonies of VS45 containing pSB1A3 were patched onto chloramphenicol plates. We set up an overnight digest of miniprepped pSBIC3 using ECORI and PSTl

Day 12 Wet lab 07/07/15

We ran an agarose gel of overnight digest, however, no bands were visible. We then set up overnight cultures of pSBIC3 and pSBIA3 to be miniprepped the next day. Finally, we set up an overnight digest of pSBIC3

Day 13 Wet lab 08/07/15

An agarose gel of the overnight digest was run, however, no bands were visible. We then did a miniprep of pSBIA3 and pSBIC3, followed by an overnight digest of these plasmids.

Day 14 Wet lab 09/07/15

We ran another agarose gel of digested pSBIA3 and pSBIC3 - no bands were visible. We then set up overnight cultures of MS349 containing pSBIA3 LIMS and MS340 containing pSBIC3, ready to be miniprepped

Day 15 Wet lab 10/07/15

We miniprepped pSBIA3 and pSBIC3, then focussed on dry lab work for the remainder of the day

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. We then set up an overnight digest of miniprepped plasmids pSBIA3 and pSBIC3 using the enzymes ECORI and PSTI

Day 17 Wet lab 14/07/15

Another agarose gel of digested plasmids pSBIA3 and pSBIC3 was set up, this time it worked. We then miniprepped the overnight cultures from the night before. We then ran a gel and carried out gel extraction of both plasmids. Following this, we ran an analytical gel using Hyperladder I in order to quantify the plasmids. Finally, we set up overnight cultures of Top10 cells containing the plasmid pVS72

Day 18 Wet lab 15/07/15

We miniprepped pVS72, then transformed it into VS45. Following the transformation, we plated the cells onto combined Chloramphenicol and Ampicillin plates. We then ran a gel of leftover digested pSBIA3 and pSBIC3 from 14/07/15. From this we were able to gel extract the plasmids.

Day 19 Wet lab 16/07/15

First, we counted the overnight colonies of VS45 with pVS72. From this, we were able to calculate 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

We produced fresh LB agar plates containing Chloramphenicol and Ampicillin. Next, our transformed VS45 with pVS72 colonies were streaked onto fresh Chloramphenicol/Ampicillin plates and incubated overnight

Day 22 Wet lab 21/07/15

We made Congo red plates with 0.2% L-Arabinose and 500ml LB broth containing 0.2% L-Arabinose. We then set up overnight cultures 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. We then prepared our cultures for TEM and AFM

Day 24 Wet lab 23/07/15

We did TEM imaging, however, our results were inconclusive.

Day 25 24/07/15

Dry lab day

Day 26 27/07/15

We transformed Top10 cells with pVS105 (negative control plasmid) and streaked it onto Ampicillin LB plates. We also transformed VS45 with pVS72 and plated it onto Chloramphenicol/Ampicillin LB plates.

Day 27 28/07/15

We made new Chloramphenicol broth and Ampicillin broth. We then used the Amp broth to resuspend Top10 cells with pVS105. We then resuspended VS45 with pVS72 in combined Chloramphenicol and Ampicillin broth. We incubated these cultures overnight at 37ºC. For the remainder of the day, we focussed on dry lab tasks.

Day 28 29/07/15

In the morning, we miniprepped the Top 10 cells containing pVS105. This will be our negative control plasmid. Following this, we diluted our culture of VS45 with pVS72 to an OD₆₀₀ of 0.1 in 3ml of LB. Once the correct OD₆₀₀ had been achieved, we spot plated 5μl of the culture onto inducing plates (containing Arabinose and IPTG), non-inducing plates and Congo red plates (both inducing and non-inducing).

Day 29 30/07/15

We transformed VS45 competent cells with the negative control plasmid (pVS105). We then plated it onto combined Chloramphenicol and Ampicillin plates.

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