IGEM:Harvard/2006/Cyanobacteria

"Required Reading"

Cyanobacteria backround and practicality

• 1. Genetic noise in the cyanobacterial oscillator
     • Very good PhD thesis by Jeffrey Chabot which has general cyanobacteria information and culturing information. Also deals with using a GFP reporter. From the vanO lab.
  2. Working with algae
     • General information on growing cyanobacteria
  3. Light conversion guide
     • Conversion information of units supplied by Peter Weigele.

Incubator/Supplies

• Incubator Dimensions: 20 x 20 in.
• Lighting: 500 foot candles of cool white fluorescent
  • A 40-watt bulb at about 15cm will provide 500 foot candles of illumination. This will fall to about 200 foot candles at 50cm.
  • Need a light meter to measure illumination.
• Shopping List:
  • 2-3 20 x __ cool white fluorescent light fixtures. 40w?
  • 20x20 plexiglass sheet
  • wire foil
  • duct tape
  • Timer (hour increments)
  • extension cord compatible with timer
  • Some way to move the brackets/shelf in the incubator

Possible Molecular Mechanisms

Activator/Repressor

• Barkai and Leibler 2000
• Modeled after Eukarayotic systems
• Probably not true
  • KaiABC vary as activator/repressor
  • Transcription/translation not essential (invitro experiment)

"Activator-repressor model from 2000, by Chabot 2005"

KaiC phosphorylation model

• Xu et al 2003
• Previous research showed
  • Cells without KaiA had all unphosphorylated KaiC
  • Cells without KaiB has all phosphorylated KaiC
  • KaiA protein constant
  • Iwasaki et al. 2002
• Note: If this model holds true than our experiment in E. coli should show some silencing of genes downstream of KaiBC? We could test it by putting a reporter right downstream of KaiBC easily... very interesting.
• Note: This would be a good question to ask someone: if we put a reporter right downstream of KaiBC what should happen to that reporter.

"KaiC phosphorylation model from 2003, by Chabot 2005"

KaiB spaitotemporal localization model

• Kitayama et al 2003
• Idea that KaiB rotates location from the membrane to cytosol
  • Doesn't the in-vitro experiment disprove this?

"KaiB spaitotemporal localization, 2003"

Transcriptional/Translational independent model

• Tomita, Nakajima et al 2005
• Minimal oscillator and an extended timing system
• Best oscillation system currently developed

"Transcriptional/translational independent, 2005"

Questions that we need to ask

• Q: If we put a reporter downstream of kaiBC what happens to it
• Q: Do we know what the sigma factor is in Kondo et al
• Q: Possible ways for reporting if oscillation works
• Q: Any other mechanisms?
• Q: How bad is the codon bias problem / would we need to actually mutate parts of the genome to move to E. coli?

Project Presentation

3000bp, 11cents/base. --> synthetic

File:Presentation.ppt

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

---

• 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.

"Image of figure shown in Xu paper"