Day 1 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 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 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 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 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 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 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 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 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 03/07/15

We calculated the transformation efficiency of pSBIA3 in VS45 cells.

Day 11 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 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 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 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 10/07/15

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

Day 16 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 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 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 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 focused on dry lab tasks.

Day 21 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 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 22/07/15

VS45 with pVS72 was plated on the Congo Red plates. We then prepared our cultures for TEM and AFM.

Day 24 23/07/15

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

Day 25 24/07/15

We used this day to focus on dry lab tasks.

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 focused 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. Whilst half of the team were working in the lab, the other half attended the London meetup at London Birkbeck.

Day 31 03/08/15

We focused on dry lab tasks such as planning the questions to ask MP's and developing the questionnaire.

Day 32 04/08/15

Our transformed pVS105 in VS45 weekend cultures were spotted onto Chl + Amp plates (both inducing and non-inducing) and Congo Red. These plates were incubated overnight at 37ºC.

Day 33 05/08/15

We checked our overnight plates were checked for contamination. Following this, we transformed pVS105 and pVS72 into VS45, plated them out and then incubated them at 37ºC overnight.

Day 34 06/08/15

We set up overnight cultures of pVS105 in VS45 and pVS72 in VS45.

Day 35 07/08/15

As the colonies did not grow overnight, we set up more colonies in the morning, with the OD₆₀₀ being checked in the evening. The transformation of pVS105 and pVS72 into VS45 was then repeated. The transformations were then plated out and incubated at 37ºC.

Day 36 10/08/15

Our miniprepped plasmids (pSBIC3, pSBIA3, pVS105 and pVS72) were digested and run on an agarose gel. VS45 colonies containing pVS105 and pVS72 were resuspended in liquid LB and cultured overnight at 37ºC.

Day 37 11/08/15

First we checked the OD of the overnight cultures. We transformed pSBIA3 and pSBIC3 into Top10 cells. We made a glycerol stock solution of pVS105 and pVS72 to store it for future use. We spot plated the pVS72 and pVS105 separately onto plates for imaging. We then miniprepped pVS72 and pVS105 from the overnight cultures.

Day 38 12/08/15

First, we re-transformed pSBIA3 into Top10 cells. We then did PCR of pSBIC3 and pVS72.

Day 39 13/08/15

As PCR did not work properly the day before, we repeated the procedure. We also scanned the streaked Congo Red plates.

Day 40 14/08/15

We purified the PCR product, then digested it. We then measured the concentrations of our stock of pSBIA3 and pSBIC3.

Day 41 17/08/15

We ligated pSBIC3 and pVS72, then transformed it into competent Top10 cells. We then plated it onto Chloramphenicol plates. We used an agarose gel to test if the digest from the day before had worked successfully and also to find out if the PCR had worked. We also prepared our samples for imaging.

Day 42 18/08/15

We set up overnight cultures of the transformed ligations.

Day 43 19/08/15

We miniprepped the overnight cultures of the ligated pSBIC3 and pVS72 that were cultured overnight. Following the miniprep, we digested the plasmids. Simultaneous to the digest, we transformed 1µl of the plasmid into VS45 competent cells.

Day 44 20/08/15

We ran an agarose gel of digested plasmids from the night before. Overnight cultures of ligations of pSBIC3 and pVS72 were also set up.

Day 45 21/08/15

We miniprepped the overnight cultures. Following the miniprep, we ligated pSBIC3 and pVS72 as the previous ligations did not work. Alongside the ligations, some of the team members used Gibson assembly to fuse fragments containing CsgAss and Sup35NM to pSBIC3. The Gibson assembly products were then transformed into competent cells and incubated at 22ºC over the weekend.

Day 46 24/08/15

We carried out Gibson assembly of fragments containing CsgAss, Sup35NM and Cytochrome b₅₆₂, followed by transformation into competent cells. The ligations from the day before were digested and run on a gel. We set up overnight cultures of the Gibson assembly product from 21/08/15.

Day 47 25/08/15

We carried out PCR of fragments and set up overnight cultures of the Gibson assembled fragments that grew overnight. We then miniprepped and set up a digest of the Gibson Assembled fragments containing CsgAss and Sup35NM from the overnight cultures. From the digest, an agarose gel was run.

Day 48 26/08/15

We did Gibson Assembly of fragments containing CsgAss and Sup35NM with pSBIA3 and Gibson Assembly of fragments containing CsgAss, Sup35NM and Cytochrome b₅₆₂. Following this, we transformed the product into competent cells. We PCR purified the product from the previous day. After PCR purification, we digested the purified product and then ran it on an agarose gel.

Day 49 27/08/15

We performed a double digest of plasmids pSBIC3 and pSBIA3, then ran it on a gel. We then set up overnight cultures of cells containing pSBIC3 and pSBIA3.

Day 50 28/08/15

We miniprepped pSBIC3 and pSBIA3 out of the overnight cultures. We then digested this and ran it on a gel.

Day 51 01/09/15

We digested pVS72 and ran it on an agarose gel. We then set up more overnight cultures of colonies containing pSBIC3 and pSBIA3.

Day 52 02/09/15

We miniprepped pSBIC3 and pSBIA3, then digested it and ran it on a gel. As the digest worked, we then carried out a gel extraction of the plasmids.

Day 53 03/09/15

We digested the gel extracted pSBIC3 and ran it on a gel to check it was the correct size. We then measured the concentration of it and then used it to do Gibson assembly with our ordered fragments. Following Gibson assembly, we transformed the product into competent cells and plated it out.

Day 54 04/09/15

Half of the team attended the London meetup, while the other half continued with the lab work. In the lab we resuspended some of the colonies from our transformed cells with the Gibson assembly product and incubated it overnight at 37ºC. We then did another Gibson assembly of the fragments into pSBIA3. The product was then transformed into competent cells. pVS72 was digested for 1 hour at 37ºC and then run on an agarose gel. In the evening, we took part in the Stacey Symposium at the University of Kent.

Day 55 05/09/15

Half of the team attended the London iGEM meetup, whilst the other half attended the University of Kent open day. In the lab, we miniprepped our overnight cultures, digested it and ran it on a gel. The pVS72 that was run on a gel the previous day was extracted. Weekend cultures of pSBIA3 and pSBIC3 were set up

Day 56 07/09/15

We transformed pSBIC3 with RFP and pSBIA3 with RFP from the iGEM distribution kit into Top10 cells. We carried out PCR on our ordered fragments, then transformed it into Top10 cells and VS45 cells. We also miniprepped pSBIA3 and pSBIC3 and digested it for one hour. Following the digest, we ran it on a diagnostic gel. A diagnostic digest and gel was carried out on both pVS72 and pVS72 with cytochrome b₅₆₂.

Day 57 08/09/15

We PCR purified the PCR products from the previous day. We then used this to do ligations with pSBIC3. We did a Gibson assembly of pSBIC3 with some of our ordered fragments. We also transformed the ligations into Top10 cells and the Gibson assembly product into competent cells. Finally, overnight cultures of pSBIC3 with RFP were set up.

Day 58 09/09/15

pSBIC3 with RFP was miniprepped out of the overnight cultures. It was then digested for 1 hour then we heat inactivated the enzymes by placing it in a waterbath at 80ºC. Ligations of pSBIC3 with PCR products were carried out and more overnight cultures of pSBIC3 in Top10 cells was set up.

Day 59 10/09/15

We miniprepped pSBIC3, digested it and then heat inactivated the enzymes. We then ligated pSBIC3 with the PCR products and did a Gibson assembly of pSBIC3 with the ordered fragments. We also made a new stock of competent Top10 cells. Overnight cultures of ligated products from 09/09/15 were set up

Day 60 11/09/15

We miniprepped the ligated pSBIC3 with PCR products from the overnight cultures. We then digested them and ran them on an agarose gel. We then set up weekend cultures at 22ºC of the Gibson assembly products.

Day 61 14/09/15

We miniprepped our plasmids, digested them and then ran it on a gel to see if we had successfully assembled our biobricks.

Day 62 15/09/15

We digested our biobricks and ran them on an agarose gel. We also prepared our samples for shipment.

Day 63 16/09/15

We digested more of our biobrick and ran it on an agarose gel. We spent the remainder of the day writing for the wiki.