ProjectStudying Synechococcus elongatus for biophotovoltaics

ProjectStudying Synechococcus elongatus for biophotovoltaicsLinks:http://www.bio.tamu.edu/synecho/index.htmlhttp://genome.jgi-psf.orghttp://genome.jgi-psf.org/synel/synel.home.htmlhttp://cyano.genome.jp/http://genome.microbedb.jp/cyanobase/SYNPCC7942

How to bioengineer a novel bio-photovoltaic system?Obtain a sequence by PCR, then clone it into a suitable plasmidTOPO allows directional cloning of PCR products!Topoisomerase I cuts at CCCTT, winds and religatesTransform product into E.coliIdentify clones by PCRConfirm by sequencingTransform into SynechococcusSequence enters genome by homologous recombination atNS1

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!

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

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 workin Synechococcus

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 workin Synechococcus•5’ primer must start CACC tobind cloning site

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 workin Synechococcus•5’ primer must start CACC tobind cloning site•Next bases should be ATG tobe optimal distance from RBS

Initiation in Prokaryotes1) IF1 & IF3 bind 30S subunit, complex binds 5' mRNA

Initiation in Prokaryotes1) IF1 & IF3 bind 30S subunit, complex binds 5' mRNA 2) Complex scans down until finds Shine-Dalgarno sequence, 16S

rRNA binds S-D

Initiation in Prokaryotes1) IF1 & IF3 bind 30S subunit, complex binds 5' mRNA 2) 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

Initiation in Prokaryotes1) IF1 & IF3 bind 30S subunit, complex binds 5' mRNA 2) Complex scans down until finds Shine-Dalgarno sequence, 16S

rRNA binds S-D3) IF2-GTP binds tRNAi

fMet

complex binds start codon

Initiation in Prokaryotes1) IF1 & IF3 bind 30S subunit, complex binds 5' mRNA 2) Complex scans down until finds Shine-Dalgarno sequence, 16S

rRNA binds S-D3) IF2-GTP binds tRNAi

fMet

complex binds start codon4) Large subunit binds

IF2-GTP -> IF2-GDPtRNAi

fMet is in P siteIFs fall off

Elongation1) EF-Tu brings charged tRNA into A site

Elongation1) EF-Tu brings charged tRNA into A site• anticodon bindsmRNA codon, EF-Tu-GTP -> EF-Tu-GDP

Elongation1) EF-Tu brings charged tRNA into A site• anticodon binds codon, EF-Tu-GTP -> EF-Tu-GDP2) ribosome bonds growing peptide on tRNA at P site to a.a. on tRNA at A site

Elongation1) EF-Tu brings charged tRNA into A site• anticodon binds codon, EF-Tu-GTP -> EF-Tu-GDP2) ribosome bonds growing peptide on tRNA at P site to a.a. on tRNA at A site peptidyl transferase is 23S rRNA!

Elongation3) ribosome translocates one codon• old tRNA moves to E site & exits• new tRNA moves to P site• A site is free for next tRNA• energy comes from EF-G-GTP -> EF-G-GDP+ Pi

Termination1) Process repeats until a stop codon is exposed2) release factor binds nonsense codon •3 stop codons = 3 RF in prokaryotes (1 RF binds all 3 stop codons in euk)

Termination1) Process repeats until a stop codon is exposed2) 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 site4) Ribosome falls apart

PoisonsInitiation: streptomycin, kanamycinElongation:• peptidyl transferase: •chloramphenicol (prok)• cycloheximide (euk)

• translocation• erythromycin (prok)• diptheria toxin (euk)

Puromycin causes premature termination