IGEM:Harvard/2006/Presentation cyano week2



Think about these questions when preparing your project proposals for the group meeting.

For each project idea:

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 * 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)
 * 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; 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
 * 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