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Selecting for Small
Small Φ group
W13 final presentation
Outline
Background Application T7 structure Genome In vivo assembly In vitro assembly
Project The big picture Recent results
Plans for spring
Background – Application
Mateu M. G., Protein Engineering, Design & Selection, 2011, 24(1–2), 53–63
Natural viruses and their capsids are not optimal.
Size is an important issue Two methods for virus
capsid studies Site directed mutagenesis Direct evolution
Background – T7 structure
Icosahedral shape ~60nm in diameter Mature capsid is 2nm thick 415 capsid proteins
90% gp10a and 10% gp10b Volume:120 x 103 nm3
Has double stranded DNA Has 39,937 base pairs Encodes for all the proteins necessary for DNA
replication Has many non-essential genes that can be removed
Background – T7 Genome
Enterobacteria phage T7
Minor capsid protein
Major capsid protein
DNA polymerase
Connector protein Assembly/scaffolding protein
Background – in vivo Assembly Capsid proteins bind the connector protein ring
(gp8) The capsid radially extends outward with the help
of scaffolding proteins (gp9) Gp9 is somehow ejected from the capsid Terminase stuffs the DNA into the capsid The capsid irreversibly expands as DNA enters The tail proteins attach
Background – in vitro Assembly
a: proheads Isolated from WT T7
b: 9-10 heads Scaffolding and head proteins
c: converted heads
Isolated from WT T7
d: 10 heads Scaffolding, head, and connector proteins
Cerritelli M. E., J. Mol. Biol., 1996, 258, 286-298
T7 capsids can be assembled in vitro utilizing plasmids Basis for site-directed mutagenesis Stability of such capsid require more research.
Project – Big Picture
Capsid in vitro assembly
Determine mutant capsid
size
Compare genome and capsid size
Compare structure subunits
Identify sites for mutation
Design primer/plasmi
d for SDM
Apply mutagen
Select for plaque
size
Determine mutant capsid
size
Determine titer
Site directed mutagenesis
Direct evolution
Project – Big Picture
Capsid in vitro assembly
Determine mutant capsid
size
Compare genome and capsid size
Compare structure subunits
Identify sites for mutation
Design primer/plasmi
d for SDM
Apply mutagen
Select for plaque
size
Determine mutant capsid
size
Determine titer
Site directed mutagenesis
Direct evolution
Project – Recent results 3/1
3/15
3/30
4/15
Start of iGem
Start of small Φ group
Select Φ for study• T7 • Qβ
Design plan of attack
SDM design for T7 and Qβ
T7 arrive!
T7 spot test and tittering experiment
Mutation design Sequence comparison Viability comparison Spot test Tittering result
Mutation Design Major/Minor Capsid Proteins
Minor capsid protein produced from ribosomal slippage in a series of T’s
Alter Genome Size Knock out DNA polymerase
Project – Recent results
Sequence comparison (partial)
Project – Recent results
57-59nm
~60nm
Basic experimental procedure
Project – Recent results
Spot test Titering test
Viability comparison Top: BL21; bottom: W3110
Project – Recent results
Spot test on BL21
Project – Recent results
T7+ T7 new
Titer result on BL21
Project – Recent results
-5 titer
-15 titer
control
-10 titer
Determine concentration of the phage with titering experiments
Direct evolution N-methyl-N’-nitro-N-nitrosoguanidine?
Site-directed mutagenesis Isolate phage genome and clone genes into plasmid
Plans for spring