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Project Studying Synechococcus elongatus for biophotovoltaics

Project Studying Synechococcus elongatus for biophotovoltaics. Project Studying Synechococcus elongatus for biophotovoltaics Links: http://www.bio.tamu.edu/synecho/index.html http://genome.jgi-psf.org http://genome.jgi-psf.org/synel/synel.home.html http://cyano.genome.jp/

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Project Studying Synechococcus elongatus for biophotovoltaics

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  1. Project Studying Synechococcus elongatus for biophotovoltaics

  2. Project Studying Synechococcus elongatus for biophotovoltaics Links: http://www.bio.tamu.edu/synecho/index.html http://genome.jgi-psf.org http://genome.jgi-psf.org/synel/synel.home.html http://cyano.genome.jp/ http://genome.microbedb.jp/cyanobase/SYNPCC7942

  3. How to bioengineer a novel bio-photovoltaic system? Obtain a sequence by PCR, then clone it into a suitable plasmid TOPO allows directional cloning of PCR products! Topoisomerase I cuts at CCCTT, winds and religates Transform product into E.coli Identify clones by PCR Confirm by sequencing Transform into Synechococcus Sequence enters genome by homologous recombination at NS1

  4. How to bioengineer a novel bio-photovoltaic system? • Obtain a sequence by PCR, then clone it into a suitable plasmid • We’re adding DNA, but want Synechococcus to make a protein!

  5. How to bioengineer a novel bio-photovoltaic system? • Obtain a sequence by PCR, then clone it into a suitable plasmid • We’re adding DNA, but want Synechococcus to make a protein! • Design primers that bind 5’ of target gene’s start codon and 3’ of stop codon so Synechococcus can translate it

  6. How to bioengineer a novel bio-photovoltaic system? • Obtain a sequence by PCR, then clone it into a suitable plasmid • We’re adding DNA, but want Synechococcus to make a protein! • Design primers that bind 5’ of target gene’s start codon and 3’ of stop codon so Synechococcus can translate it • pSyn1 provides promoter, • Ribosome Binding Site & • terminator sequences that work • in Synechococcus

  7. How to bioengineer a novel bio-photovoltaic system? • Obtain a sequence by PCR, then clone it into a suitable plasmid • We’re adding DNA, but want Synechococcus to make a protein! • Design primers that bind 5’ of target gene’s start codon and 3’ of stop codon so Synechococcus can translate it • pSyn1 provides promoter, • Ribosome Binding Site & • terminator sequences that work • in Synechococcus • 5’ primer must start CACC to • bind cloning site

  8. How to bioengineer a novel bio-photovoltaic system? • Obtain a sequence by PCR, then clone it into a suitable plasmid • We’re adding DNA, but want Synechococcus to make a protein! • Design primers that bind 5’ of target gene’s start codon and 3’ of stop codon so Synechococcus can translate it • pSyn1 provides promoter, • Ribosome Binding Site & • terminator sequences that work • in Synechococcus • 5’ primer must start CACC to • bind cloning site • Next bases should be ATG to • be optimal distance from RBS

  9. Initiation in Prokaryotes 1) IF1 & IF3 bind 30S subunit, complex binds 5' mRNA

  10. Initiation in Prokaryotes IF1 & IF3 bind 30S subunit, complex binds 5' mRNA Complex scans down until finds Shine-Dalgarno sequence, 16S rRNA binds S-D

  11. Initiation in Prokaryotes • IF1 & IF3 bind 30S subunit, complex binds 5' mRNA • Complex scans down until finds Shine-Dalgarno sequence, 16S rRNA binds S-D • Next AUG is Start codon, must be w/in 7-13 bases

  12. Initiation in Prokaryotes IF1 & IF3 bind 30S subunit, complex binds 5' mRNA Complex scans down until finds Shine-Dalgarno sequence, 16S rRNA binds S-D IF2-GTP binds tRNAifMet complex binds start codon

  13. Initiation in Prokaryotes • IF1 & IF3 bind 30S subunit, complex binds 5' mRNA • Complex scans down until finds Shine-Dalgarno sequence, 16S rRNA binds S-D • IF2-GTP binds tRNAifMet complex binds start codon • Large subunit binds • IF2-GTP -> IF2-GDP • tRNAifMet is in P site • IFs fall off

  14. Elongation 1) EF-Tu brings charged tRNA into A site

  15. Elongation • EF-Tu brings charged tRNA into A site • anticodon binds mRNA codon, EF-Tu-GTP -> EF-Tu-GDP

  16. Elongation • EF-Tu brings charged tRNA into A site • anticodon binds codon, EF-Tu-GTP -> EF-Tu-GDP • 2) ribosome bonds growing peptide on tRNA at P site to a.a. on tRNA at A site

  17. Elongation • EF-Tu brings charged tRNA into A site • anticodon binds codon, EF-Tu-GTP -> EF-Tu-GDP • 2) ribosome bonds growing peptide on tRNA at P site to a.a. on tRNA at A site • peptidyl transferase is 23S rRNA!

  18. Elongation • 3) ribosome translocates one codon • old tRNA moves to Esite & exits • new tRNA moves to P site • A site is free for next tRNA • energy comes from • EF-G-GTP -> EF-G-GDP+ Pi

  19. Termination • 1) Process repeats until a stop codon is exposed • 2) release factor binds nonsense codon • 3 stop codons = 3 RF in prokaryotes (1 RF binds all 3 stop codons in euk)

  20. Termination • 1) Process repeats until a stopcodon is exposed • 2) release factor binds nonsense codon • 3 stop codons = 3 RF in prokaryotes (1 RF binds all 3 stop codons in euk) • 3) Releases peptide from tRNA at P site • 4) Ribosome falls apart

  21. Poisons • Initiation: streptomycin, kanamycin • Elongation: • peptidyl transferase: • chloramphenicol (prok) • cycloheximide (euk) • translocation • erythromycin (prok) • diptheria toxin (euk) • Puromycin causes premature termination

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