Team:Kent/Notebook
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.
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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
- Bacteria that deals with free radicals
- 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
- Solar panels
- 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
- What are the key proteins? Can we obtain them?
- Can it be incorporated into E.coli
- What are the applications.
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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.
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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.
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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
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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.
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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.
