iGEM 2015 — Team Kent

# Team:Kent/Notebook

iGEM Kent 2015

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# Notebook

[June](#day1)
[July](#day8)
[August](#day31)
 [September](#day51)

### Day 1 *22/06/15* - It All Begins!

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

![](https://static.igem.org/mediawiki/2015/2/23/Team_Kent_Notebookphoto1.jpg)

### 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 pSB1C3 out of MS348.

### Day 4 *25/06/15*

We set up an overnight digest of pSB1C3 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 pSB1C3 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* - The First of Many Gels

We checked the plates that had been inoculated 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.

![](https://static.igem.org/mediawiki/2015/f/ff/Team_Kent_CompetentCells.jpg)

### 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 pSB1C3, ready for gel extraction the following day.

### Day 7 *30/06/15*

We miniprepped pSB1A3 and set up an overnight digest of it. We then ran the overnight digest of a large quantity of pSB1C3 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 pSB1C3 ready to be extracted, we then set up a digest of our entire stock of pSB1C3. 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 pSB1C3.

### Day 9 *02/07/15*

On this day we miniprepped pSB1C3 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.

![](https://static.igem.org/mediawiki/2015/e/e0/Team_Kent_JamesPractical.jpg)

### Day 10 *03/07/15*

We calculated the transformation efficiency of pSB1A3 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 pSB1C3 using EcoRI and Pstl.

### Day 12 *07/07/15* - or The Day We Realized We Hated Gels

We ran an agarose gel of overnight digest, however, no bands were visible. We then set up overnight cultures of pSB1C3 and pSB1A3 to be miniprepped the next day. Finally, we set up an overnight digest of pSB1C3.

### Day *08/07/15*

An agarose gel of the overnight digest was run, however, no bands were visible. We then did a miniprep of pSB1A3 and pSB1C3, followed by an overnight digest of these plasmids.

![](https://static.igem.org/mediawiki/2015/9/92/Team_Kent_stacysym1.jpg)

### Day 14 *09/07/15*

We ran another agarose gel of digested pSB1A3 and pSB1C3 - no bands were visible. We then set up overnight cultures of MS349 containing pSB1A3 LIMS and MS340 containing pSB1C3, ready to be miniprepped.

### Day 15 *10/07/15*

We miniprepped pSB1A3 and pSB1C3, then focused on dry lab work for the remainder of the day.

### Day 16 *13/07/15*

Overnight cultures of MS349 containing pSB1A3 LIMS and MS340 containing pSB1C3 were set up, ready to be miniprepped. We then set up an overnight digest of miniprepped plasmids pSB1A3 and pSB1C3 using the enzymes ECORI and PSTI.

![](https://static.igem.org/mediawiki/2015/6/66/Team_Kent_igem76.jpg)

### Day 17 *14/07/15*

Another agarose gel of digested plasmids pSB1A3 and pSB1C3 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 pSB1A3 and pSB1C3 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.

![](https://static.igem.org/mediawiki/2015/6/62/Team_Kent_igem91.jpeg)

### 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* - Hello Congo Red!

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.

![](https://static.igem.org/mediawiki/2015/2/22/Team_Kent_igem45.jpg)

### 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* Only a small setback

We used this day to focus on dry lab tasks.

![](https://static.igem.org/mediawiki/2015/5/5d/Team_Kent_igem47.jpeg)

### 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).

![](https://static.igem.org/mediawiki/2015/d/d6/Team_Kent_igem4.jpg)

### 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* - London Meetup

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.

![](https://static.igem.org/mediawiki/2015/5/56/Team_Kent_igem69.jpg)

### 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.

![](https://static.igem.org/mediawiki/2015/d/d6/Team_Kent_igem42.jpg)

### 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* - Waiting game

Our miniprepped plasmids (pSB1C3, pSB1A3, 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 pSB1A3 and pSB1C3 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.

![](https://static.igem.org/mediawiki/2015/8/80/Team_Kent_plateart.png)

### Day 38 *12/08/15*

First, we re-transformed pSB1A3 into Top10 cells. We then did PCR of pSB1C3 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 pSB1A3 and pSB1C3.

![](https://static.igem.org/mediawiki/2015/9/96/Team_Kent_workshop_2.jpeg)

### Day 41 *17/08/15*

We ligated pSB1C3 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 pSB1C3 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.

![](https://static.igem.org/mediawiki/2015/1/1d/Team_Kent_stacey_5.jpeg)

### Day 44 *20/08/15*

We ran an agarose gel of digested plasmids from the night before. Overnight cultures of ligations of pSB1C3 and pVS72 were also set up.

### Day 45 *21/08/15*

We miniprepped the overnight cultures. Following the miniprep, we ligated pSB1C3 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 pSB1C3. 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 *b*562, 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.

![](https://static.igem.org/mediawiki/2015/f/fd/Team_Kent_afmimage.png)

### 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 pSB1A3 and Gibson Assembly of fragments containing CsgAss, Sup35NM and Cytochrome *b*562. 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 pSB1C3 and pSB1A3, then ran it on a gel. We then set up overnight cultures of cells containing pSB1C3 and pSB1A3.

![](https://static.igem.org/mediawiki/2015/c/c1/Team_Kent_igem35.jpg)

### Day 50 *28/08/15*

We miniprepped pSB1C3 and pSB1A3 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 pSB1C3 and pSB1A3.

### Day 52 *02/09/15*

We miniprepped pSB1C3 and pSB1A3, then digested it and ran it on a gel. As the digest worked, we then carried out a gel extraction of the plasmids.

![](https://static.igem.org/mediawiki/2015/d/df/Team_Kent_img201509.jpg)

### Day 53 *03/09/15*

We digested the gel extracted pSB1C3 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* - UK Meetup Day 1& Stacey Symposium

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 pSB1A3. 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* - UK Meetup Day 2

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 pSB1A3 and pSB1C3 were set up

### Day 56 *07/09/15*

We transformed pSB1C3 containing an RFP insert as well as pSB1A3 with RFP from the iGEM distribution kit into Top10 cells. We carried out PCR on the fragments we had ordered, then transformed them into competent Top10 cells and VS45 cells. We also miniprepped pSB1A3 and pSB1C3 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 *b*562.

![](https://static.igem.org/mediawiki/2015/c/c4/Team_Kent_igem46.jpeg)

### Day 57 *08/09/15*

We PCR purified the PCR products from the previous day and then used this to do ligations with pSB1C3. We did a Gibson assembly of pSB1C3 with our ordered fragments. We also transformed the ligations into Top10 cells and the Gibson assembly product into competent cells. Finally, overnight cultures of pSB1C3 with RFP were set up.

### Day 58 *09/09/15*

pSB1C3 with RFP was miniprepped out of the overnight cultures. It was then digested for 1 hour with PstI and EcoRI, then we heat inactivated the enzymes by placing it in a waterbath at 80ºC. Ligations of pSB1C3 with PCR products were carried out and more overnight cultures of pSB1C3 in Top10 cells was set up.

### Day 59 *10/09/15*

We miniprepped pSB1C3, digested it and then heat inactivated the enzymes. We then ligated pSB1C3 with the PCR products and did a Gibson assembly of pSB1C3 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

![](https://static.igem.org/mediawiki/2015/2/27/Team_Kent_crane.jpg)

### Day 60 *11/09/15* - Hello Heme!

We miniprepped the ligated pSB1C3 with PCR products from the overnight cultures. They were then digested and ran on an agarose gel. We then set up weekend cultures at 22ºC of the Gibson assembly products. A heme plate was inoculated with VS45 cells expressing the fusion protein CsgA  ss  - Sup35NM - Cyt  *b*   562  and incubated over the weekend.

### Day 61 *14/09/15*

We miniprepped our plasmids, digested them and then ran them on a gel to see if we had successfully assembled our biobricks. We set up two more soft agar plates, one containing VS45 expressing the fusion protein CsgA  ss  - Sup35 and the other with VS45 containing no external plasmid. We incubated these at 37ºC overnight. We measured the conductivity of the inoculated plates produced on the 11/09/15. We also used AFM imaging to confirm that our fusion protein was being expressed in the incubated VS45 cells

### Day 62 *15/09/15*

We digested our biobricks and ran them on an agarose gel. We also prepared our samples for shipment. We measured the conductivity of our three different soft plates, one expressing conductive amyloid fibres, one expressing amyloid fibres lacking cytochrome and our negative control, with no amyloid. AFM imaging was done to see the progress of amyloid growth on inducing plates

![](https://static.igem.org/mediawiki/2015/4/4d/Team_Kent_gibsonfrag.jpg)

### 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. We measured the conductivity of our soft plates. AFM imaging was done to see expression of amyloid fibres in our cells

### Day 64 *17/09/15*

We used AFM to image our cells. We also measured the conductivity of the soft plates. The rest of the time was spent writing for the wiki and updating it.

### Day 65 *18/09/15* - "The Cold Never Bothered Me Anyways"

Today is Wiki Freeze Day! Everyone has been hard at work proofreading and making our wiki perfect.

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