Team:Kent/Notebook

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Pre - June

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Pre-June

February

  • Team Kent for iGEM 2016 was formed.
  • We had our first iGEM meeting where we got acquainted with some of the team members.
  • We also met with our team supervisor
  • The iGEM timeline was discussed and key dates highlighted.
    • October 27-31 – iGEM Jamboree
    • Submissions deadline – 21 st October 2016
    • October 19 – Wiki-freeze
  • We discussed our aims and goals for our project.
  • Specific medal criteria – Bronze, Silver, Gold
  • A team organiser and social media officer was appointed.

March

  • Our iGEM team was complete 10 students, 2 supervisors and 3 advisors.
  • We completed our official iGEM registration individually and as a team.
  • We began establishing our plans for the summer.
    • We were appointed a space in the bioscience lab.
    • We would have weekly meeting from the start of our summer projects.
  • We began brainstorming for our project.
    • Problem based approach - What problems needed to be addressed?
    • Are we inventing something novel? Are we improving something that has already been done?
    • How can we approach a problem that has already been addressed to get a different response?
    • Is this project feasible in the time available?
    • Is there a sensible approach for validation?
    • How will we explore this project?
  • We viewed the track selections for iGEM
    • Which track would our project lay in?
  • Tracks
    • Diagnostics
    • Energy
    • Environment
    • Food and nutrition
    • Information processing
    • Manufacturing
    • Therapeutics
    • Art and design
    • Hardware
    • Measurement
    • Software
  • We began exploring previous iGEM projects by looking at previous team wikis.
  • Our ideas
    • Bacterial Batteries
    • Magnetic bacteria
      • To conduct energy
      • Combat heavy metal poisoning
      • Lead
    • CRISPR – specific alteration of genomes
    • Material formation – with applications to fibre formation (continuation of nanowire project) (fibre formation an area of Dr Xue’s expertise)
    • Magnetic nanowires to conduct electricity?

April

  • The brainstorming continued.
    • Bacteria that deals with free radicals
      • Fluorescent proteins that detect the radicals
    • Fluorescent proteins
      • For detection
    • Bacteria which grows hair
      • Clear applications to this
    • Swimming bacteria
    • Protein cages for drugs (be transported through Tat pathway)
    • Protein sculptures – visual
  • We re-visited the idea of magnetic bacteria.
  • We started researching magnetic bacteria species.

May

  • We continued brainstorming ideas
    • Solar panels
      • Harnessing photosynthetic capability of bacteria
      • Phylum cyanobacteria (slow)
      • Researchers are thinking of modifying the bacteria
      • Current problems:
        • Removal of waste material (solar panel practically inaccessible after installation)
      • Efficiency of energy conversion, only 8% when solar panel gives 40%
      • Evolved to protect themselves from full sunlight, hence, low efficiency
    • Bacteria antimicrobial peptides (protein targeting)
      • To combat infections
      • Here, the protein is the drug
    • Bacteria preventing the growth of other bacteria
  • Exam season!
  • After months of brainstorming it was time to make a decision.
  • We revisited an idea we had been discussion from our first brainstorming session.
  • Magneto bacteria
    • It has been ‘tricked’ to bio-mineralizing crystals is which iron was replace with other metals
    • By growing the bacteria in a solution with a high concentration of both metals
    • Shown that it can incorporate manganese into the crystal structure of magnetite
    • Some success with cobalt
    • Uses: waste removal or drug delivery
  • We started researching relevant literature relating to this topic
    • What are the key proteins? Can we obtain them?
    • Can it be incorporated into E.coli
    • What are the applications.
  • We created a google drive where would feed in information throughout our project.

June

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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?

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.

July

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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,
  • 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.

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’
  • 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

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

August

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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.
  • 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.

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

  • 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

September

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September

August 29 th – September 4 th (Week 10)

Day 45

  • The AFM imaging of samples commenced.
  • 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)

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.
  • 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.

October

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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

  • 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.
  • 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:





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