Team:Kent/Notebook


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

  • 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

    Day 60 11/09/15

  • Miniprep of ligated pSBIC3 with PCR products
  • Digest of the minipreps
  • Agarose gel of the digests
  • Day 61 14/09/15

  • Miniprep of our plasmids
  • Digest of our plasmids
  • Diagnostic Agarose gel to see if we have our biobricks