IGEM:Harvard/2006/Cyanobacteria
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3000bp, 11cents/base. --> synthetic
Think about these questions when preparing your project proposals for the group meeting.
For each project idea:
- What is the specific goal of the project?
- Populate Biobricks
- Biobrick KaiABC oscillator (for use in either cyanobacteria AND/OR e. coli)
- [1] Shows the location of kaiA,BC in WH8102 strain. 2.866kb for kaiABC + non-coding region.
- Research shows that KaiABC show oscillation independently (Nakajima et al. 2005)
- Test the oscillator in E. coli to create a "nightlight"
- Use a luciferase gene reporter, which was done in (Kondo et al. 2000)
- Also can measure KaiC activity; create a chimeric protein w/GFP
- Synthesis of ~3kb KaiABC w/ codon replacement of Ala of Leu to use in E. coli
- .11/bp w/o error correction; $2/bp with error correction (Tian et. al 2004)
- But the Church lab has a better way of doing this?
- Provides backup in case direct movement of KaiABC into E. coli fails
- Codon bias problem with 2 amino acids (can't find source but I found it the other day): then, we can synthetically modify the codons for these 2 aa's to be compatiable in e. coli
- .11/bp w/o error correction; $2/bp with error correction (Tian et. al 2004)
- Alternate phrasing, courtesy of Kit Parker - what is the "deliverable?" The thing you will point to and say "this is our project?"
- Our deliverable is a (multiple?) BioBrick part(s)
- What are two or three possible means of implementing the idea?
- Biobricks the cyanobacteria KaiABC
- Implement directly into E. coli to create a "nightlight"
- Synthesis of a E. coli compatible KaiABC and implement in E. coli
- Create a circuit with other BioBricks
- Last resort: Just create a cyanobacteria "nightlight" if all E. coli steps fail
- Risk
- How many untested things have to work for the project to succeed?
- Should work unless something in E. coli causes it not to
- Reporter gene should have no problem
- Codon bias may be a problem
- If more proteins are involved than KaiABC
- It worked in vitro however...
- Transcription regulation of the kaiABC proteins
- We know that KaiA mRNA constant as KaiC fluctuates (Wang et. al 2005)
- Should work unless something in E. coli causes it not to
- How will you test whether those things work or not?
- If we don't get results / alternative methods such as synthesis
- How will you adjust your plan when one of these things fails to work?
- We have backup plans, such as only implementing a "nightlight" in cyanobacteria
- How will you minimize the time/effort/resources lost to a failed design?
- Can your time/effort/resources apply to more than one design simultaneously?
- How many untested things have to work for the project to succeed?
- Reward
- How cool, fun, exciting is the project for you?
- What if any is the usefulness or societal benefit of the project?
- Clock oscillator
- Can experimentally vary it from 14h to 40h (Kondo et. al 2000) based on point mutations
- Can further discretise by half
- A bacterial "timer"
- Unlikely, but a computer syncronization method (but too slow...)
- Metal detector
- Stun gun
- nightlight
- Clock oscillator
- What is going to impress the judges in November?
- Biobricks part!
- Timeline
- What are the project milestones? (design, construction, testing)
- Getting WH8102 strain of cyanobacteria 1-2 wks
- Prof. Wang at Yale wrote a review, so he may know - will contact him
- Otherwise may have take field trip to tour Japan or check papers for sources
- Creating a cyanobacteria biobrick / extracting KaiABC genes 1-2 wks
- Designing primers can be done beforehand
- Designing a feasible E. coli version of KaiABC 1-2 wks
- Reseach into the necessary modifications
- Making the modifications of the 3kb sequence (should be fast)
- Send to synthesize
- Implementing into E. coli both versions Long time (5wk+)
- Design either chimeric protein or luciferase (Perry?)
- Implementation and testing
- What is the estimated time required for each? (always overestimate)
- If you can't reach your ultimate goal by August, is there a satisfying intermediate goal?
- We WILL create a biobricked part that works for cyanobacteria at least
- And if worse comes to worse we'll make a cyanobacteria nightlight
- What is the immediate next step in pursuing the project?
- See first 3 before
- If DNA synthesis will be required, how soon will you have the sequence designed?
- 1-2 weeks
- What are the project milestones? (design, construction, testing)
- Nakajima et. al: in vitro, the proteins oscillate abeit not with as large of an amplitude