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