A Synthetic Biology Approach to Discerning Circadian Output Pathways in Cyanobacteria

1. A Synthetic Biology Approach to Discerning Circadian Output Pathways in Cyanobacteria Zhipeng Sun MCB186 Final Project December 13, 2006

2. The Kai Clock in Cyanobacteria • KaiC autophosphorylates and dephosphorylates • KaiA promotes phosphorylation • KaiB inhibits KaiA • Transcription-translation independent • Period: 14-60 h A B C P P P P P P b Phosphorylation of KaiC output Time Nakajima et al. (2005)

3. Question • By what molecular mechanism does KaiC phosphorylation drive clock output? A B P P ? C PpsbAI::luxAB reporter SasA? RpaA? Hypothesis: SasA-RpaA complex as intermediary (Takai et al. 2006) Hypothesis: SasA-RpaA complex as intermediary (Smith and Williams 2006)

4. 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) Synechococcus PCC7942 E. Coli ECNR1 http://www.igmors.u-psud.fr/images/Coli.gif http://www.abc.net.au/science/news/img/environment/cyanobacteria220805.jpg

5. 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)

6. Step 1: Reconstitute transcription pathway • Clone RNAP α1α2, β, β‘, ω, σ subunits into vector containing lac promoter + RBS +antibiotic • PubMedEntrez Protein entries for PCC7942: • 24 sigma factors • 1 ea. α1α2, β, β‘ subunits • Lambda-red recombination into E. coli, antibiotic selection lac vector α1α2, β, β‘, ω, σ Cat/Kan Lambda-red Recombination E. Coli genome Selection and screening LB-MIN+MARKER Plate E. Coli colony

7. Step 1: Reconstitute transcription pathway E-coli RNAP • Clone and transform PsbAI::luxAB from cyanobacteria lac α1α2, β, β‘, ω, σ Transcription/translation CyanoRNAP PpsbAI psbAI luxAB • Verification • Bioluminescence should be constant over time • Western blot with anti-cyanobacteria-RNAP antibody output

8. Step 2: Reconstitute circadian control • Clone and transform KaiA, KaiB, and KaiC under inducible e. coli promoter to express in 1:1:4 ratio by molar mass Verification Western Blot KaiC over circadian cycle Nakajima et al. (2005) • 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)

9. Proof of PrincipleKai Proteins Interact in E. coli A A P P P P P P P P P C B C C Hsieh, Lau, Ramos, and Sun (unpublished, 2006)

10. Step 3: Screen for pathway components • Reconstitute SasA and RpaA and observe bioluminescence • Horizontal gene transfer from cyanobacteria to e. coli until bioluminescence is oscillatory • Selectively knockout genes until bioluminescence is constant • Repeat 2-3 for other proteins • Label e. coli DNA with DAPI and visualize over circadian cycle to test Smith and Williams (2006) hypothesis Verification A B C P P output

11. Conclusions In 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

12. References Thanks for listening, and thanks for a great semester! Court 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 NatlAcadSci 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 cyanobacterialKaiCphosphorylation 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 cyanobacteriumSynechococcuselongatus. Proc NatlAcadSci 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 NatlAcadSci U S A 2006 Aug 8; 103(32) 12109-14.