IGEM:Harvard/2006/Cyanobacteria: Difference between revisions
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*Modeled after Eukarayotic systems | *Modeled after Eukarayotic systems | ||
[[Image: Barkai_Leibler.jpg | thumb | Activator-repressor model]] | [[Image: Barkai_Leibler.jpg |thumb| "Activator-repressor model from 2000"]] | ||
===Possible Project Ideas=== | ===Possible Project Ideas=== |
Revision as of 11:37, 20 June 2006
Possible Molecular Mechanisms
Activator/Repressor
- Barkai and Leibler 2000
- Modeled after Eukarayotic systems
Possible Project Ideas
People to contact
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 catalog
- Biobrick a KaiABC oscillator (for use in cyanobacteria AND/OR e. coli)
- This site shows the location of the kaiABC genes in WH8102 strain. 2.866kb for kaiABC + non-coding region.
- Research shows that the KaiABC proteins alone will oscillate in vitro (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
- Estimated cost is $0.11/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?
- DNA synthesis provides a backup in case direct insert of KaiABC into E. coli fails
- There is a known 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 compatible in e. coli
- Estimated cost is $0.11/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 BioBrick part(s)
- What are two or three possible means of implementing the idea?
- Biobrick the cyanobacteria KaiABC
- Insert directly into E. coli to create a "nightlight"
- Synthesize 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
- More proteins may be involved than KaiABC
- But KaiABC have been shown to work in vitro
- Transcription regulation of the KaiABC proteins
- We know that KaiA mRNA remains 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?
- It's cool, fun, AND exciting!
- What if any is the usefulness or societal benefit of the project?
- Clock oscillator
- Can experimentally vary the period of the oscillator from 14h to 60h (Kondo et. al 2000) with KaiC point mutations
- Can further discretise by half
- A bacterial "timer"
- Could be used as a clock for gene circuits, analogous to a clock signal in silico (but may be too slow)
- Nightlight
- Clock oscillator
- What is going to impress the judges in November?
- Biobricks part!
- How cool, fun, exciting is the project for you?
- 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 how to obtain this strain - we will contact him
- Otherwise we may have to take a field trip to tour Japan, or check papers for sources
- EDIT: Strain PCC7942 works also; MIT says it is the model system for studying circadian rhythm; [1] has the location for KaiA, B, C. Will email people for these two strains.
- Creating a cyanobacteria biobrick / extracting KaiABC genes 1-2 wks
- Designing primers can be done beforehand
- Designing a feasible E. coli version of KaiABC (can be done simultaneously with step 1) 1-2 wks
- Research 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
- Getting WH8102 strain of cyanobacteria 1-2 wks
- 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 steps 1 and 3 above
- 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 KaiABC proteins oscillate robustly by themselves, albeit with a lower amplitude than in vivo
Don't deleteFrom Xu et al 2004, they have anti-KaiC polyclonal antibodies which they used to do a phosphorylated KaiC assay; we can use this method to analyse KaiC fluctuation.
From the paper:
KaiC Phosphorylation Assay. Cyanobacterial cultures were grown to an OD750 of 0.2. After a 12-h dark pulse, the cultures were treated with 10�MIPTGfor 3 h at 30°C in light with air bubbling, and�40 ml of the cultures were collected for preparation of total extracts. Immunoblot analysis for KaiC was performed on 5 �g of total proteins per lane according to the previous description (20), except that we used a highly specific mouse polyclonal antibody to KaiC and a SuperSignalWest Pico Chemiluminescent Substrate (Pierce). The phosphorylated KaiC signals were quantified as before (16) and analyzed by Student’s t test.