A Synthetic Biology Approach to Discerning Circadian Output Pathways in Cyanobacteria

Zhipeng SunMCB186 Final ProjectDecember 13, 2006

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The Kai Clock in Cyanobacteria•KaiC autophosphorylates and dephosphorylates•KaiA promotes phosphorylation•KaiB inhibits KaiA•Transcription-translation independent•Period: 14-60 h

Time

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f Kai

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Nakajima et al. (2005)

Question

• By what molecular mechanism does KaiC phosphorylation drive clock output?

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SasA? RpaA?

PpsbAI::luxABreporter

Hypothesis: SasA-RpaA complex as intermediary (Takai et al. 2006)

Hypothesis: SasA-RpaA complex as intermediary (Smith and Williams 2006)

Synthetic Approach: Pathway Reconstitution in E. coli

• Traditional CoIP vs. proposed reconstitution• Tools– Lambda-red recombination (Court et al. 2002)– Conjugation (Li and Elledge 2006)– Cloning– In-vitro transcription (Melton et. al 1984)

http://www.igmors.u-psud.fr/images/Coli.gifhttp://www.abc.net.au/science/news/img/environment/cyanobacteria220805.jpg

Synechococcus PCC7942

E. Coli ECNR1

Cyanobacteria PCC6803 genome cloned into B. subtilis

•“Megacloning,” or mass horizontal gene transfer

•Cyanobacteria rRNA operon genes induced cell death

•Resulting cell contained cyanobacteria mRNA transcripts

Itaya et al. (2005)

Step 1: Reconstitute transcription pathway

1. Clone RNAP α1α2, β, β‘, ω, σ subunits into vector containing lac promoter + RBS +antibiotic– PubMed Entrez Protein entries for PCC7942:

• 24 sigma factors• 1 ea. α1α2, β, β‘ subunits

2. Lambda-red recombination into E. coli, antibiotic selection

Cat/Kanvector

E. Coli genome

Selection and screening

LB-MIN+MARKER PlateE. Coli colony

α1α2, β, β‘, ω, σ

Lambda-red Recombination

lac

Step 1: Reconstitute transcription pathway

3. Clone and transform PsbAI::luxAB from cyanobacteria α1α2, β, β‘, ω, σ

E-coli RNAP

CyanoRNAP

psbAI

PpsbAI

luxAB

output

Transcription/translation

lac

Verification•Bioluminescence should be constant over time•Western blot with anti-cyanobacteria-RNAP antibody

Step 2: Reconstitute circadian control

1. Clone and transform KaiA, KaiB, and KaiC under inducible e. coli promoter to express in 1:1:4 ratio by molar mass

VerificationWestern Blot KaiC over circadian cycle

2. Compare KaiC expression through anti-KaiC Western Blot:• With KaiA, B, C under e. coli promoter• With KaiA, B, C under cyanobacteria promoter(If the expression level is similar, do not put subsequent cyanobacteria

genes under e. coli promoters to save time)

Nakajima et al. (2005)

Proof of PrincipleKai Proteins Interact in E. coli

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Hsieh, Lau, Ramos, and Sun (unpublished, 2006)

Step 3: Screen for pathway components

1. Reconstitute SasA and RpaA and observe bioluminescence

2. Horizontal gene transfer from cyanobacteria to e. coli until bioluminescence is oscillatory

3. Selectively knockout genes until bioluminescence is constant

4. Repeat 2-3 for other proteins5. Label e. coli DNA with DAPI

and visualize over circadian cycle to test Smith and Williams (2006) hypothesis

Verification

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output

ConclusionsIn case of failure…• Change promoters and RBS

to inducible or low expression

• Change promoter system from cyanobacteria to e. coli

• Use different reporter systems from cyanobacteria (eg. PKaiBC)

• Create a cDNA library from extracted cyanobacteria genes and conduct microarray analysis

Versatility of Approach• Can be modified for other

pathways in cyanobacteria– Light-input pathway– Biochemistry of phase shift

and resetting• Can be genearlized for

simple prokaryotes• Can be automated

ReferencesCourt DL, Sawitzke JA, and Thomason LC. Genetic engineering using

homologous recombination. Annu Rev Genet 2002; 36 361-88. Hsieh H, Lau J, Ramos D, Sun Z. Reconstitution of the cyanobacteria

oscillator in E. coli. iGEM 2006, unpub.Itaya M, Tsuge K, Koizumi M, and Fujita K. Combining two genomes in

one cell: stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome. Proc Natl Acad Sci U S A 2005 Nov 1; 102(44) 15971-6.

Li MZ and Elledge SJ. MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules. Nat Genet 2005 Mar; 37(3) 311-9.

Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, and Green MR. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 1984 Sep 25; 12(18) 7035-56.

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.

Smith RM and Williams SB. Circadian rhythms in gene transcription imparted by chromosome compaction in the cyanobacterium Synechococcus elongatus. Proc Natl Acad Sci U S A 2006 May 30; 103(22) 8564-9.

Takai N, Nakajima M, Oyama T, Kito R, Sugita C, Sugita M, Kondo T, and Iwasaki H. A KaiC-associating SasA-RpaA two-component regulatory system as a major circadian timing mediator in cyanobacteria. Proc Natl Acad Sci U S A 2006 Aug 8; 103(32) 12109-14.

Thanks for listening, and thanks for a great semester!