1. II.
Condi)onal
mutant
strain
design
and
screening
CreS
non-essential
MreB
essential
Crea)ng
condi)onal
mutants
of
essen)al
genes
Jan
Clement
San)ago,
Anastasiya
Yakhnina,
Zemer
Gitai
Molecular
Biology
Department|
Princeton
University
|
Princeton,
NJ
08544
Abstract
Methods
Preliminary
Results
References
Acknowledgements
Creating deletion strains is a well-established method to study gene function by
enabling direct phenotypic comparisons of wild-type cells and deletion mutants.
However, the usefulness of deletions as a genetic tool is limited to genes not essential to
the organism’s survival. Here, we’ve designed a convenient method for constructing
conditionally lethal depletion mutants of essential genes found in Caulobacter
crescentus in order to screen them for cell shape defects in a high-throughput fashion.
We created a genetic system where the endogenous gene of interest is tagged with ssrA,
which encodes a short peptide degradation tag, and introduced another copy of that
gene under the control of a xylose promoter. Thus, the gene of interest is effectively
expressed only in the presence of xylose. For a high-throughput generation of such
depletion strains, each gene is cloned by an in vivo LR reaction from a Caulobacter
Gateway entry vector library into destination vectors encoding a xylose promoter and an
in-frame ssrA tag. This process allows for significantly faster and more efficient vector
construction. As a test of principle, we tried depleting two genes whose loss-of-function
effects are known: mreB, an essential gene required for normal cell width; and creS, a
non-essential gene whose deletion causes Caulobacter to lose its curvature. This
method can potentially be applied to other systems to study loss-of-function phenotypes
of all essential genes that are otherwise inaccessible to study via deletion.
recipient cells in Kan & Rif media
I.
Expression
vector
construc)on
Our
lab
developed
an
in
vivo
variaBon
of
the
Invitrogen
Gateway®
cloning
system
to
allow
for
efficient
vector
construcBon
in
a
high-­‐throughput
fashion.
gene A
ssrAP xyl
gene A
Kanr
no xylose
no xylose
with xylose
with xylose
• TradiBonal
deleBons
cannot
be
done
on
essenBal
genes
,
but
condiBonal
mutants
can
be
created
that
deplete
these
genes
under
certain
condiBons
• Many
structural
genes
determining
cell
shape,
size,
length,
width,
etc,
are
essenBal
• Exactly
480
Caulobacter
essenBal
genes
have
been
idenBfied
out
of
3,763
total
genes
(12.75%)
• There’s
only
liZle
informaBon
on
what
role
most
of
these
genes
play
in
cell
shape/
structure
• Our
lab
has
an
ORFeome
library
of
all
C.
crescentus
genes,
each
in
individual
Gateway
®
compaBble
vectors
• This
allows
for
rapid,
high-­‐throughput
screening
to
study
the
effects
of
all
essenBal
genes
on
cell
shape
Rifr
ccdBs
Kanr
Rifr
ccdBs
Possible plasmid products:
Rifr
ccdBs
via conjugation
chromosome
gene A gene A
in presence of xylose
Kan
r
degraded protein product
Caulobacter crescentus (ΔPxyl)
in 96-well plates with xylose
media
(automated) imaging; screen
for cell shape defects
culture overnight, wash,
grow on few hours without
xylose
homologous
recombination
wt Δ creS
MreB
depletion
Christen, B., Abeliuk, E., Collier, J. M., Kalogeraki, V. S., Pasarelli, B., Collier J. A.,
Fero, M.J., McAdams, H.H., Shapiro L. (2011) The essential genome of a bacterium.
Molecular Systems Biology, 7: 528,. doi: 10.1038/msb.2011.58
Werner, J.N., Chen, E.Y., Guberman, J.M., Zippilli, A.R., Irgon J.J., Gitai, Z. (2009).
Quantitative genome-scale analysis of protein localization in an asymmetric
bacterium. Proceedings of the National Academy of Sciences, 106(19), 7858-7863.
doi: 10.1073/pnas.0901781106
wt
Ø Both showing ΔcreS phenotype; creS not being expressed at all, with or
without xylose!
Ø Both showing wt phenotype!
Ø Several cultures of mutant mreB didn’t grow even with xylose
Ø Only those that grew are pictured here, presumably wt for mreB that
somehow survived selection
Ø Probably mreB was not expressed in mutants, which died since mreB is
essential
Ø Possiblilitiy: since att site is also translated, it could have interfered with
protein folding
Why
do
this?
Why
Caulobacter
crescentus?
It’s
a
curved,
rod-­‐shaped,
asymmetric
bacterium
with
two
disBnct
poles,
which
makes
it
easy
to
study
and
track
changes
in
bacterial
cell
shape
and
structure.
I would like to acknowledge Princeton Summer Undergraduate Research Program and
its sponsors, Princeton Molecular Biology Department, Lewis-Sigler Institute of
Integratative Genomics and Howard Hughes Medical Institute, for the research
opportunity this summer. I’m thanking the lab members of my host lab, my principal
investigator Zemer Gitai, and most of all my adviser Anastasiya Yakhnina for their
advice, time, and tireless support for this project.
attR1 attR2
destination
vector
Cut and ligate
in ssrA, a peptide signal
for proteasomal
degradation
P
xyl
CC 0001
entry vector
CC 0002
CC 0003
attL1 attL2
Gateway compatible C. crescentus
ORFeome library
Kan
r
In vivo LR reaction
+Xis
+Int
ccdBr
destination vector
host cell
entry vector host cell
conjugations
recipient cell
Rifr
ccdBs
ccdB toxin
“Gateway”
cassette