IGEM:Harvard/2006/Cyanobacteria

"Required Reading"

Cyanobacteria backround and practicality

1. Genetic noise in the cyanobacterial oscillator

• 1. • 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.

Papers on latest findings

1. Nakajima M, Imai K, Ito H, Nishiwaki T, Murayama Y, Iwasaki H, Oyama T, and Kondo T. Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science. 2005 Apr 15;308(5720):414-5. DOI:10.1126/science.1108451 |

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"