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Team:Imperial College London/Notebook/week2
From 2011.igem.org
Diary
This is our diary page which records the daily activities of the team. Click on the links below to see a summary of events and activities happening each week.
Week 2: 11th July to 17th July
Monday, 11th July
Action points: Development of wiki, Brainstorming, DNA assembly talk, Brainstorming Presentation of the ideas Day. With the sad news that James is still in hospital we started to work again. After a meeting with the advisors on Friday, we were now ready to brainstorm again about many more ideas. In the morning we went to Queens Lawn to enjoy a bit of sunshine to help us thinking. After a while some of the ideas were formed. At noon we had a talk about DNA assembly and a after a quick lunch we split into groups and continue developing our ideas (you can find them on the brainstorming page). Before finishing for the day, we all presented some of the points and ideas, discarded some and kept some for further investigation. Tomorrow we hope to see James among us and brainstorm some more.
DNA assembly presentation:
DNA assembly can kill projects if done incorrectly. It takes around 3 days for 1 assembly: Day 1: Start cultures Day 2: Assemble Day 3: Screen (might take more than one day).
Assembly day: Miniprep ~30 mins. Extract part ~75-135 mins. Purify part ~90 mins. Assemble (make flanking sequences single stranded and mix and anneal) ~75-135 mins. Transform ~60 mins. ~6.5 hours! That is being optimistic!
Two techniques: Short Overlap (BioBricks)- Maximum two parts at the same time. Long overlap (Gibson/User) - Safely assemble 5 parts, theoretically 10.
Short: Miniprep --> Restriction digest --> Gel extract --> Ligate--> Transform All parts submitted must be in this format. Can use the parts from miniprep a maximum of around 4 times. ~6 bp overhang. Low Tm (~10 degrees celsius). Ligase works best at 37 degrees celsius though. Need to compromise between both these parameters.
Long: Miniprep --> PCR --> DpnI digest + purify or gel extract --> Assembly reaction--> Transform PCR primer could work first time or take weeks to get to work due to possibilities of non-specific binding and other errors that can occur with an inadequate primer. Gibson ~40bp overhang while User ~20bp overhang. User require a uracil for every 7th base. Uracils are expensive bases to buy. Gibson still work for DNA fragments down to ~91bp. Need to be careful that they're not too much shorter.
Part insulation: If 2 ORF's are next to each other one might transcribe into another. Solve through three methods: 1: Place a terminator in between. Problem is that if there are too many terminators with the same sequence, deletions might occur. Check Biofab IS website for different terminators. 2: Face parts away from each other. 3: Spacing them away from each other. However, read-through is very long.
Reusable parts: BioBricks allows us to reuse parts easily. Can this be done with the Long overhang methods? Prefix and suffix attached to GOI. Use linkers. Depending on linker we attach one GOI to another in whatever orientation and order we want.
Libraries: Three types: Mutants, Mutagenomics and Rational design. Sources: Other labs/registries, epPCR, degenerate synthesis.
Tuesday, 12th July
Today we continued brainstorming and came up with a few new ideas but had to scrap a few that are not feasible.
1. Spores & rain
a. Need an application, not just making rain
b. Empty virus capsid instead of spore?
2. Mucin on plants
a. On surface of leaves, yeast could produce mucin coat if it gets very hot to avoid water loss
b. How does light interact with mucin? Could have opposite, heating effect.
3. Eutrophication
a. Aerobic bacterium that turns nitrate and ammonia into N gas.
b. Special lipids prevent poisonous intermediate chemicals from killing the cells because reaction compartmentalized
c. Very slow growth rate (2 weeks doubling time)
d. Can increase growth rate or express enzymes in E. Coli
e. About 10 enzymes involved in the pathway; could just express a few
f. How many enzymes? Can they be expressed in E. Coli?
4. Waste water
a. Estrogen compounds
b. Coupling a sensor to degradation process
c. Lacases can degrade one of the compounds. Not sure about how to target the others
d. Bio-sequestration?
5. Anti-freeze protein in plants
a. Need license with bacteria to make tumours
6. pH sensor and regulator
a. Bacteria balance internal pH but not really external
b. Moss can turn pH of water acidic in bogs.
c. Alkaline?
d. Huge problem of acidity in the ocean – crustaceans cant build shells
7. Solar cell
a. Bacterial rhodopsin?
b. Hydrogenase and nitrogenase
8. Nematodes
a. Secrete volatile organic compounds from stable bacteria
b. Olfactory chemotaxis to attract nematodes
c. Need parasitic nematode specific killing – research this
d. Silence genes with RNAi by feeding E. coli?
9. Mining bacteria
a. Metal binding proteins
b. Bacterial catalyst?
10. Termite enzyme
a. Symbiosis between nematode and termite to convert wood cellulose into sugar
Wednesday, 13th July
We had more discussion and brainstorming with the people from RCA. We came up with various themes so as to help us in the generation of ideas. More new ideas were produced and some of yesterday's ideas were further developed. We came up with the following list of 8 ideas at the end of the day. 1) Bacterial solar cell 2) Auxin-secreting bacteria 3) Termite enzymes converting wood to sugar 4) Degradation of Polycyclic Aromatic Hydrocarbon 5) Bacteria-targeting nematodes 6) Oestrogenic endocrine disrupters in waste water 7) Filling in the green gap 8) Anammox bacteria to treat eutrophication Among these, the most promising and favourite ones are idea 2, 3 and 5. We will continue to focus and developed on these ideas. As for the rest of the ideas, we might look further into them. But if nothing can be developed from there, we will discard them and come up with more new ideas.
Thursday, 14th July
Day 9 Entry (14th/July/2011)
Morning:-
- More brainstrorming
- Came up with the following 9 ideas: Chris - Termites: turning cellulose into sugar; Frank - Auxin-secreting bacteria; Si - dodecane into primary alcohol; Nick - photosynthetic bacteria and ATP production; Ming - PHA into PAH; Rebekka - treatment of oestrogen in waste water; Nikki - nematodes; Nina & Yuanwei - cellulose breakdown library with the enzyme from rabbit cecum Hamiltonian path problem and network alignment
- Finally, Nina and Yuanwei's ideas were shut down since: 1. The cellulose-breakdown idea is the same as the termites one. 2. The Hamiltonian path and DNA computing idea is definitely worth thinking about to solve the protein-protein interaction problem, but it is too unrealistic for us.
Afternoon:-
- Presentation! Smooth and generally satisfying.
- Two ideas survived out of seven: Termites group: Chris, Rebekka, Si, Yuanwei and Nick; Auxin group: Frank, Ming, Nikki and Nina.
- Drink at union bar: Who is the next model for Ming ? Frank ? Goat and dude - naughty :] Yuanwei - we will get you drunk next time.
Evening:-
- Dinner at Oriental Canteen.
- All of us went to the common room in James' hall to prepare for the presentation tomorrow.
- Presentation key points: 1. Title 2. Problem: background introduction 3. Project description 4. Previous iGem projects 5. Genetic circuits 6. Modelling 7. Experiments 8. Human practice 9. Safety , security and ethics.
Friday, 15th July
Today, we prepared presentations on the two selected project proposals to the professors and Claire and Susanna from LSE.
After much back and forth and cake, we decided that project auxin was superior to project termite. Happiness ensued.
