iGEM 2016 — Team Kent

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# Team:Kent/Notebook

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  + [★ Measurement](https://2016.igem.org/Team%3AKent/Measurement)
  + [★ Model](https://2016.igem.org/Team%3AKent/Model)

# Notebook

---

## This page contains the documentation for our Mag-nano-tite Project.

## It will be split into 6 sections containing the work done through the duration of our project.

![](https://static.igem.org/mediawiki/2016/1/1e/TeamKentNotebookPre-June.jpeg)

## Pre - June

[Click here](#season1)

**X**

# Pre-June

---

![](https://static.igem.org/mediawiki/2016/8/88/TeamKentNotebookJune.jpeg)

## June

[Click here](#season2)

**X**

# June

---

## May 31- June 5

* We began to gather scientific papers (PubMed, WebofKnowledge, Science Direct) to post on to
  our shared folder.* We explored the iGEM archives for similar ideas identify Bio-bricks (Mam proteins?)* Identify key words to search magnetosomes, MMS6 protein, nano-particles.

## June 6 - June 12

* We began to plan how we would conduct our experiments.
  + What genes are relevant?+ Can we obtain them+ Do we need specific strains of E. coli?+ Can we purchase these strains?+ If not who do we speak to, to obtain these strains?+ What safety risks do these pose?

![](https://static.igem.org/mediawiki/2016/e/e7/TeamKentGDriveMagnetosomes.jpeg)

## June 13 - June 19

* We began to obtain sequences we would use.* It was important here to start to identify the experiments we were going to use.

## June 20 - June 26

* We continue reading through and understanding the relevant literature.* We began to understand our safety implications of our project.

![](https://static.igem.org/mediawiki/2016/3/36/TeamKentNotebookJuly.jpeg)

## July

[Click here](#season3)

**X**

# July

---

## June 27 th – 3 rd July (Week 1)

## Day 1

* We received our lab induction. We received training on how to use unfamiliar equipment.
* Having already researched the different protocols we would be using during our project we conducted risk assessments.
* We received training on the correct use and disposal of waste and materials.
* We were appointed different plasmids:
  + pET3a mamX/mamT/mamP
  + pET23B SecS mamX/ mamT/ mamP
  + pSB1A3 RFP
  + pEC86
  + pSB1c3 RFP
  + pET3a
  + pSB1C3
* We began writing our project descriptions for the upcoming deadline.
* We set up our social media accounts: Twitter,
* Facebook and Instagram.

## Day 2

* We made LB plates and broth ensuring to add the relevant antibiotics (ampicillin or chloramphenicol).

## Day 3

* Parts from our distribution kits were re-suspended.
* Our plasmids were transformed in top10 cells and left overnight for 15-18hours incubation.

## Day 4

* Re-streaks were performed on successful transformations.
* Unsuccessful transformations were repeated.
* The writing of our project was continued.
* Registration for a student account for Enthought Canopy,
![](https://static.igem.org/mediawiki/2016/d/db/TeamKentJulyImage1.jpeg)* Tutorials were completed on TutorialSpoint/Python
* We attempted to ‘scrape’ one iGEM wiki

## July 4 th – July 10 th (Week 2)

## Day 5

* Liquid cultures were prepared frim our transformed cells.
* Using SnapGene we carried out plasmid map analysis using Snapgene to identify compatible restriction sites on our individual plasmids.

## Day 6

* Our plasmids were purified from the overnight liquid cultures.
* The concentration of the DNA of our individual plasmids were determined.
* Restriction enzyme digests were conducted based on the restriction sites identified on the plasmid maps including the appropriate buffer.
  + pET3a – Xbal, HindIII, promega E
  + pET3a mamP – Xbal, EcoRI, promega H
  + pET3a mamT – Xbal, HindIII, promega E
  + pET3a mamX – Xbal, HindIII, promega E
  + pET23b mamP – NdeI, EcoRV, promega D
  + pET23b mamT – XbaL, PstI, promega H
  + pET23b mamX – NdeI, EcoRV, promega D
  + pET23b mamP – NdeI, EcoRV, promega D
  + pEC86 – Ndel, EcoRV, promega D

## Day 7

* Agarose gels were conducted to validate our various plasmids.
* We uploaded our project description onto our wiki
* Gels were imaged.

## Day 8

* Troubleshooting began who electrophoresis gels which were unsuccessful.

## Day 9

* pSB1C3 RFP and pSB1A3 RFP plasmids were validated.
![](https://static.igem.org/mediawiki/2016/2/2e/TeamKentJulyImage2.jpeg)

## July 11 th – July 17 th (Week 3)

## Day 10

* Gel extraction protocols were used to purify validated plasmids.
* Liquid cultures were prepared to form glycerol stocks from validated plasmids.
* We prepared our outreach presentations for Simon
* Langton Grammar School for Boys Canterbury.

## Day 11

* We successfully gave outreach presentations at Simon
* Langton Grammar School for Boys Canterbury.
* Two presentations delivered to physics and biology classes respectively.
* Questions from both physics classes were documented.

## Day 12

* We continued with the validation of our individual plasmids with the aim to validate all our plasmids.
* Gel electrophoresis gels were continued.
* Plasmids pET3a and pET23b sec.S mamT/ mamP/ mamX were validated.

## Day 13

* Plasmids pET3a and pET23b sec.S mamT/ mamP awere transformed into BL21 cells.
* Liquid cultures were prepared from the Top10 constructs of Plasmids pET3a and pET23b SecS mamT/ mamP

## Day 14

* Glycerol stocks were made for pet3aMamT and psb1c3, pSB1C3 RFP and pSB1A3 RFP.
* Liquid cultures were made for these plasmids
* Plasmid purification was done for the necessary plasmids to build up the DNA stocks

## July 18 th – July 24 th (Week 4)

## Day 15

* Liquid cultures from BL21 cells containing SecS.MamX/MamT/MamP plasmids.
* Using SnapGene we looked at primer sequences in preparation for PCR.
* We began brainstorming for the iGEM meet up at the University of Westminster on August 17-19 th

## Day 16

* We gathered detailed the sequences of the Mam proteins using their respective SnapGene
* We ensure that we validated the sequences UniProt data and found the correct open reading frames.
* We began growing up liquid cultures in preparation for the purification of our proteins MamP and MamT.

## Day 17

* We finished gathering possible primer sequences for our Mam proteins.
* We began protein preparations for protein expression and purification.
* MamP, MamX and MamT grown in

## Day 18

* Buffers were prepared.
  + Lower Tris buffer prepared using: 1.5M Tris, 0.4% SDS, pH 8.8
  + Upper Tris buffer prepared using: 1M Tris, 0.4% SDS, pH 6.8
  + Tris-Glycine running buffer prepared – 30g Tris base, 144g Glycine, 150ml SDS (1.5% w/v)
* Liquid cultures from BL21 cells were made from transformed MamX, MamT and MamP cells and incubated overnight.

## Day 19

* SDS page gels was made.
* Samples taken from the liquid cultures were boiled to denatured protein.
* Samples obtained were run on an SDS-page gel.
* The gel was stained with comassie blue over the duration of the weekend.

## July 25 th – July 31 st (Week 5)

## Day 20

* We decided on the team name ‘Mag-nano-tite’
![](https://static.igem.org/mediawiki/2016/1/19/TeamKentJulyImage3.jpeg)* A collaboration was formed between Team Kent and UC Davis
* We wrote code for a search bar on iGEMMatch.org
* SDS PAGE gel de-stained using methanol on shaker for ~3 hours.
* Gel was imaged and proved unsuccessful.
* Troubleshooting began.

## Day 21

* Continued work on ideas/concepts for the upcoming Westminster presentation and poster.
* Nickel column B and wash buffers were made and set to the correct pH.
* overnight liquid
* Liquid cultures were also prepared from SecS.MamX, MamP, MamT and pET3a Top10 cells.

## Day 22

* The plasmids were purified from the liquid cultures made the previous day and the concentrations determined
* We contacted the iGEM team at the University of Westminster team regarding the meet up and continued work to the presentation.

## Day 23

* we conducted an inventory to determine which plasmids still required transformation, or repeats.
* pEC86 was transformed into BL21 cells.

## Day 24

![](https://static.igem.org/mediawiki/2016/9/95/TeamKentJulyImage4.jpeg)

* We attempted co-transformation into BL21 cells using plasmids secS mamP &amp; pec86) and incubated over the weekend.

![](https://static.igem.org/mediawiki/2016/e/e3/TeamKentNotebookAugust.jpeg)

## August

[Click here](#season5)

**X**

# August

---

## August 1st – August 7th (Week 6)

## Day 25

* We began preparing for the iGEM deadline on August 12th.
* We began compiling information for our abstract
* Co-transformed plasmids pEC86 + SecS MamP were made into BL21 DE3 competent cells.
* SecS MamP were transformed in BL21 in DE3 competent cells.

## Day 26

* SecS MamX were transformed in BL21 in DE3 competent cells.
* We created a code which measures the frequency of a key word for our Topic modelling programme.

## Day 27

* Competent cells were created for pEC86 and SecS MamX were, measured into 0.5mL aliquoted and stored in the -80°C deep freezer.
![](https://static.igem.org/mediawiki/2016/0/0c/TeamKentAugustImage1.jpeg)* We received our primers,

## Day 28

* The primers were re-suspended.
* We carried out our first PCR using the Mam genes (SecS and insoluble) to amplify our DNA.
* The PCR products were purified.

## Day 29

* An electrophoresis gel was conducted to validate the PCR products. This gel was unsuccessful, and the PCR was repeated adjusting the temperatures in a bid to correct the errors from the first PCR.
* Prepared Mam proteins were analysed using an SDS-page gel. They were stained using coomasie-blue for the duration of the weekend

## August 8th – August 14th (Week 7)

## Day 30

* SDS PAGE gels were de-stained; resulting image shown below.
* The team delivered a mock presentation in preparation for Westminster conference.
* Final adjustments were made for iGEM deadline for track Selections and project abstract

## Day 31

* Mam proteins were isolated using His-tag purification.
* After a third failed attempt to gain the correct PCR products, PCR was repeated using different enzymes.
* Plasmid maps were view to select new primers. primers.
![](https://static.igem.org/mediawiki/2016/b/bf/TeamKentAugustImage2.jpeg)

## Day 32

* pET3a plasmid validated.
* We had a meeting with team and supervisors to discuss direction of our project and set targets to achieve Gold medal criteria
* PCR was repeated using new primers

## Day 33

* We began researching the literature relating to the chemical synthesis of magnetic nano-particles.
* We spoke to a chemical expert in the field of nano-particles.
* We liaised with teams for a possible collaboration.

## Day 34

* iGEM deadline met, abstract submitted and track selection completed – New Applications.
* We continued sourcing the materials for the chemical synthesis of chemical nano-particles.
* Preparation for the Westminster meet-up continued.

## August 15th – August 21st (Week 8)

## Day 35

* We began working on an outline for our wiki page.
* A Gibson assembly was performed and fragment sup35nm was re-suspended.
* We finalised our presentations and posters for the University of Westminster conference.

## Day 36

![](https://static.igem.org/mediawiki/2016/2/23/TeamKentAugustImage3.jpeg)

* We started identifying improvements we could make of the previous years’ project.
* Liquid culture was prepared from pEC86 + MamP and MamT
* We observed that the induced liquid culture when spun down was red in colour likely due to the cytochrome C domain in the protein binding iron.

## Day 37

* We developed our Topic modelling programme to read multiple text files.
* Members of the iGEM team presented our poster at the University of Westminster iGEM conference
* Using nickel ion chromatography, we purified out MamX, MamT, MamP proteins

## Day 38

* Members of the iGEM team gave a spoken presentation at the University of Westminster iGEM conference

## Day 39

* We began compiling our data, results in preparation for our wiki.

## August 22nd – August 28th (Week 9)

## Day 40

* We reflected on the presentations given at the Westminster
* Gibson assemblies were performed.
* We double digested pSB1C3 and ran a gel to validated,
* We transformed NEB 5-alpha competent E. coli cells with 2ul of the Gibson reaction mixture.

## Day 41

* Colonies from plates were Re-streaked some white colonies and made liquid cultures.

## Day 42

* Re-streaks showing white colonies were mini-prepped.
* QIAprep protocol to purify the plasmids.

## Day 43

* We focused on dry lab tasks such as planning the mock questions for future presentations.

## Day 44

* We continued compiling information the wiki
* Planned future experiments

![](https://static.igem.org/mediawiki/2016/5/52/TeamKentNotebookSeptember.jpeg)

## September

[Click here](#season6)

**X**

# September

---

## August 29 th – September 4 th (Week 10)

## Day 45

* The AFM imaging of samples commenced.
![](https://static.igem.org/mediawiki/2016/0/00/TeamKentSeptemberImage1.jpeg)* Double digest of PSB1C3 RFP with Sup35nm using Ecor1 and Pst1
* PCR using usual protocol (with double digested template DNA) and original long primer on pET23B SecS genes.
* Prepared 500ml LB in 2L Baffled flasks for protein expression

## Day 46

* Repeated agarose gels
* New forward primers arrived therefore PCR was done using normal protocol (with annealing temp at 52 degree and double digested template DNA).
* We set up 4ml overnight liquid cultures containing double antibiotics, 2 for each Pet23b Sec.S MamT, MamP, and MamX with pEC86 using the same colony.
* Made fresh antibiotic samples.

## Day 47

* Made 1ml glycerol stocks using 500ul of liquid culture for Sec.S MamP, MamT and MamX with Pec86 in BL21’s.
* Using the remaining 3.5ml to inoculate 500ml LB in the Baffled flasks.
* We grew up the cells and induced with IPTG for protein expression.
* Ligation and transformations carried out.

## Day 48

* We finalised our plans for collaboration with UEA over skype conference. It was agreed that we would carry out some AFM images of their S. oneidensis cells on a variety of different surfaces.
* SDS gels carried for MamP SecS, MamX SecS, MamT SecS and stained overnight.

## Day 49

* De-strained SDS page gels and imaged them.
* In preparation, we shipped off AFM sample discs to UEA in Norwich in preparation for collaboration the following week.

## September 5 th - September 11 th (Week 11)

![](https://static.igem.org/mediawiki/2016/a/ac/TeamKentSeptemberImage2.jpeg)

## Day 50

* We prepared liquid cultures with all Mam pET23B genes.
* Using plasmid maps we designed new primers.

## Day 55

* Mini-prepped liquid cultures and ran undigested verification gels
* Re-attempted PCR
* Web design progress for our wiki.
* We used the absorbance reading at 280nm and extinction coefficient 8.73 mM -1 cm -1 to estimate the concentration of mam P as 27.4 µM.
* We used the absorbance reading at 280 and extinction coefficient 27.32 mM -1 cm -1 to estimate the concentration of mam P as 39.2 µM

## Day 56

* Using SnapGene plasmid map sequences we designed new SecS primers
* We carried out spin columns for protein purification of mamP/T/X
* Using the absorbance reading at 280 nm and extinction coefficients, we estimated the concentrations for mamP/X/T for subsequent anaerobic reaction vessels.
![](https://static.igem.org/mediawiki/2016/2/2e/TeamKentSeptemberImage3.jpeg)* Carried out the reaction using purified proteins with iron solutions within anaerobic reaction vessel.

## Day 57

* Reactions between purified proteins and iron solutions were placed onto EM grids and prepared for imaging (in vivo).

## September 12 th – September 18 th (Week 12)

* The team logo and banner were designed for review by the team.
* We collaborated UEA to AFM image with samples.
* We continued making our final constructs.

## September 19 th - September 25 th (Week 13)

* The team logo and banner were finalised.
* We continued writing
* AFM imaging of our samples continued.

![](https://static.igem.org/mediawiki/2016/1/18/TeamKentNotebookOctober.jpeg)

## October

[Click here](#season4)

**X**

# October

---

## September 26th – October 2nd

* Semester resumes
* We began planning our outreach presentation for the University of Kent summer student Bioscience Symposium.
* We prepared a presentation using updated information about our project and updated results
* We continued imaging our samples to validate our constructs, these constructs include: pSB1A3-[AraC- pBAD]-[CsgA- SS-Sup35NM- 1-61], pSB1A3-[AraC- pBAD]-[CsgA- SS-Sup35NM- 1-61- Cytb562], pSB1A3-[AraC- pBAD]-[CsgA- SS-Sup35NM]

## October 3rd – October 9th

![](https://static.igem.org/mediawiki/2016/0/02/TeamKentOctoberImage1.jpeg)

* We continued writing up our results and compiling information for our wiki.
* The team attended a University of Kent Bioscience open day. We spoke to the public about our iGEM project, the iGEM competition and its future applications.
* We continued the AFM imaging of samples.
* The final construct of pSB1A3 mamOPXT transformed.

## October 10th – October 16th

* pSB1A3 mamOPXT
* We continued the AFM imaging of our samples.
* E. coli cells were induced containing the mamOPXT plasmid, we did this by growing the cells in a liquid media containing arabinose, IPTG and Ferric Citrate.
* Electron microscopy grids prepped for imaging.
![](https://static.igem.org/mediawiki/2016/3/34/TeamKentOctoberImage2.jpeg)* Writing for wiki continued.

## October 17th – October 23rd

* The team Completed content for our wiki
* Wiki freeze.
* Final electron microscopy images produced.
* Prepared our posters, banner and presentation for the jamboree
* Outreach at second University of Kent open day.

## October 24th – October 31st

* iGEM Jamboree in Boston.

Document the dates you worked on your project.

##### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

##### Inspiration

You can see what others teams have done to organize their notes:

* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

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