1. Selecting
for Small
Small Φ group
W13 final
presentation

2. Outline
 Background
 Application
 T7 structure
 Genome
 In vivo assembly
 In vitro assembly
 Project
 The big picture
 Recent results
 Plans for spring

3. 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

4. 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

5.  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

6. 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

7. 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
Figure 1. Gel electrophoresis of purified proteins, intact heads, and dissociated heads. a, Head protein and scaffolding
protein purified after expression individually from the cloned genes, and c, 10-heads isolated after co-expression of
connector, scaffolding and head protein in the same cell, 9-10 heads isolated after co-expression of scaffolding and head
proteins in the same cell, and proheads and converted heads isolated from wild-type T7 lysates were analyzed by
polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. b, The same set of heads, without
disruption, were analyzed by electrophoresis through a 1% agarose gel (pH 8.0).
of the purified proteins into prohead shells.
Therefore, we expressed and purified these proteins
individually from the cloned genes, as described in
Materials and Methods.
The purified scaffolding protein eluted at a
position corresponding to a molecular mass of
49 kDa upon gel filtration chromatography in
buffered 50 mM NH4Cl, whereas the size calculated
Figure 2. Electron micrographs of
proheads, converted heads, 9-10
heads, and 10-heads. Samples of a,
proheads, b, 9-10 heads, c, converted
heads, and d, 10-heads, described in
more detail in the legend to Figure
1, were stained with 2% uranyl
acetate and examined by conven-
tional transmission electron mi-
croscopy (the bar represents
100 nm). The small round particles
apparent in the 10-heads, and to a
lesser extent in the converted heads,
are believed to represent a cellular
component that bands at approxi-
mately the same position as con-
verted heads in the CsCl step
gradients used. These particles do
not have the characteristic appear-
ance of T7 connectors (see Cerritelli
& Studier, 1996) and the 10-heads
contained little if any gp8
(Figure 1c).
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.

8. 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/plasmid
for SDM
Apply
mutagen
Select for
plaque
size
Determine
mutant
capsid size
Determine
titer
Site directed
mutagenesis
Direct
evolution

9. Project – Big Picture
Site directed
mutagenesis
Direct
evolution

10. 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

11.  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

12.  Sequence comparison (partial)
Project – Recent results
57-59nm
~60nm

13.  Basic experimental procedure
Project – Recent results
Spot test Titering test

14.  Viability comparison
 Top: BL21; bottom: W3110
Project – Recent results

15.  Spot test on BL21
Project – Recent results
T7+ T7 new

16.  Titer result on BL21
Project – Recent results
-5 titer
-15 titer
control
-10 titer

17.  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