IGEM:Harvard/2006/Cyanobacteria

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
  • 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)
  • 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?
  • 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
  • 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 - 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
      1. 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

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• Nakajima et. al: in vitro, the proteins oscillate abeit not with as large of an amplitude