IGEM:Harvard/2006/Presentation cyano week2

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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
    • 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)
    • 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?
  • 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
    • What is going to impress the judges in November?
      • Biobricks part!
  • Timeline
    • What are the project milestones? (design, construction, testing)
      1. 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.
      2. Creating a cyanobacteria biobrick / extracting KaiABC genes 1-2 wks
        • Designing primers can be done beforehand
      3. 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
      4. 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 steps 1 and 3 above
      • If DNA synthesis will be required, how soon will you have the sequence designed?
        • 1-2 weeks