1. Project
Studying Synechococcus elongatus
for biophotovoltaics

2. Project
Studying Synechococcus elongatus
for biophotovoltaics
Links:

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 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

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 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)
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