Background
Performing the mutagenesis
Next steps
Final Remarks
Site-directed mutagenesis of cell division proteins
for contraction generation in vitro
Don Praveen Amarasinghe
MOAC Doctoral Training Centre
University of Warwick
28th May 2013
References
Background
Performing the mutagenesis
Contents
1
Background
2
Performing the mutagenesis
3
Next steps
4
Final Remarks
Next steps
Final Remarks
References
Background
Performing the mutagenesis
Next steps
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References
Bacterial cell division
Figures reproduced from Ghigo et al. [2] and Vicente and Lowe [12]
Background
Performing the mutagenesis
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References
Three Z-ring proteins
This project focuses on three proteins known to be involved in the
formation of the Z-ring [3, 6–8, 11].
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Performing the mutagenesis
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References
Three Z-ring proteins — FtsZ
This project focuses on three proteins known to be involved in the
formation of the Z-ring [3, 6–8, 11].
Background
Performing the mutagenesis
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References
Three Z-ring proteins — ZipA
This project focuses on three proteins known to be involved in the
formation of the Z-ring [3, 6–8, 11].
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Three Z-ring proteins — ZapA
This project focuses on three proteins known to be involved in the
formation of the Z-ring [3, 6–8, 11].
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Performing the mutagenesis
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Aim of this project
The aim of this project is to tag FtsZ, ZipA and ZapA.
This is with a view to observing these proteins interact in an
“artificial cell”, enabling us to determine their role in the
mechanism of Z-ring contraction.
References
Background
Performing the mutagenesis
Contents
1
Background
2
Performing the mutagenesis
3
Next steps
4
Final Remarks
Next steps
Final Remarks
References
Background
Performing the mutagenesis
Next steps
Why (site-directed) mutagenesis?
Final Remarks
References
Background
Performing the mutagenesis
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Why (site-directed) mutagenesis?
We could tag the protein by expressing it with a fluorescent
tag attached (e.g. GFP) . . .
References
Background
Performing the mutagenesis
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Final Remarks
Why (site-directed) mutagenesis?
We could tag the protein by expressing it with a fluorescent
tag attached (e.g. GFP) . . .
. . . but these proteins are bulky.
Protein
Number of residues
GFP
238
FtsZ
383
ZipA
328
ZapA
109
References
Background
Performing the mutagenesis
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Why (site-directed) mutagenesis?
An alternative approach — tag the cysteine residues using
their thiol groups (see Kim et al. [4] for example).
O
O
H2N
H2N
OH
OH
SH
S
Tag
References
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Why (site-directed) mutagenesis?
An alternative approach — tag the cysteine residues using
their thiol groups (see Kim et al. [4] for example).
O
O
H2N
H2N
OH
OH
SH
S
Tag
If we cannot find an appropriate side-chain in a suitable place,
instigate a mutation to provide one.
Background
Performing the mutagenesis
Mutation positions
No mutations in ZapA.
Next steps
Final Remarks
References
Background
Performing the mutagenesis
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Mutation positions
No mutations in ZapA.
Four mutations in each of FtsZ and ZipA chosen.
References
Background
Performing the mutagenesis
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References
Mutation positions
No mutations in ZapA.
Four mutations in each of FtsZ and ZipA chosen.
7 out of 8 of these are Gly or Ala to Cys.
H2N
CH
C
O
O
O
OH
H2N
CH
C
OH
H2N
HS
CH
C
CH2
H
CH3
Glycine
Alanine
Cysteine
GGA
GGC
GGG
GGT
GGA
GGC
GGG
GGT
TGC
TGT
OH
Background
Performing the mutagenesis
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References
Mutation positions
No mutations in ZapA.
Four mutations in each of FtsZ and ZipA chosen.
7 out of 8 of these are Gly or Ala to Cys.
H2N
CH
C
O
O
O
OH
H2N
CH
C
OH
H2N
HS
CH
C
OH
CH2
H
CH3
Glycine
Alanine
Cysteine
GGA
GGC
GGG
GGT
GGA
GGC
GGG
GGT
TGC
TGT
Other mutation — phenylalanine residue coded for upstream
of the ZipA gene in pET-52b plasmid.
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Performing the mutagenesis
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References
Primer Design
QuikChange1 primer calculator used to generate primers (protocol
used by others [1, 10]).
1
Trademark of Stratagene / Agilent Technologies.
Background
Performing the mutagenesis
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References
Primer Design
QuikChange1 primer calculator used to generate primers (protocol
used by others [1, 10]). Basic principles behind primer building:
Need the primer size to be just right.
Too short - cannot overcome
energy costs of mismatch
1
Too long and GC rich - melting temperature too high.
Trademark of Stratagene / Agilent Technologies.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Primer Design
QuikChange2 primer calculator used to generate primers (protocol
used by others [1, 10]). Basic principles behind primer building:
Online tool to see if primers will self-anneal because of
repeated subsequences.
2
Trademark of Stratagene / Agilent Technologies.
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Performing the mutagenesis
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Protocol summary I
Methylated DNA
plasmids
Forward primer
with mutation
Reverse primer
with mutation
plus free nucleotides,
DNA polymerase and buffer
Dpn1 Digest
PCR
Mixture of plasmids with and
without mutation
37°C
1 hour
Original methylated DNA
plasmids digested by Dpn1
References
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Performing the mutagenesis
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Protocol summary II
Grow cells
Mini-prep
Transform Top10 E. coli cells
Top10 cells replicate and produce
many copies of the mutated plasmid
Harvest the mutationcontaining plasmids
References
Background
Performing the mutagenesis
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Protocol summary III
Induce cells
Grow cells
Transform over-expression strain
E. coli cells
Clones of cells and plasmids produced
Add IPTG
Incubate at 37°C for 4 hours
Cells contain protein with
desired mutation.
References
Background
Performing the mutagenesis
Results . . . ?
Mutagenesis PCR is tricky!
Next steps
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References
Background
Performing the mutagenesis
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Results — DNA gels
DNA gels were carried out to indicate the presence of PCR
product.
FtsZ - Gly to Cys at residue 21
Phusion DNA polymerase
61.7
GeneRuler Ladder
66
68.5
70
Annealing Temp / °C
References
Background
Performing the mutagenesis
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Results — DNA gels
Most gels looked like this: . . .
ZipA - Ala to Cys at residue 193
Phusion DNA polymerase
GeneRuler Ladder
Annealing
Temp / °C
ZipA - Ala to Cys at residue 328
Phusion DNA polymerase
50.0
51.7
52.8
54.3
56.0
57.4
58.5
60.0
References
Background
Performing the mutagenesis
Results — DNA gels
. . . which meant a lot of this:
Next steps
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References
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Performing the mutagenesis
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Results — DNA gels
. . . which meant a lot of this:
We did the transformations anyway. Result — lots of colonies!
References
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Results — DNA Sequence Validation
Sequencing results of transformations that produced colonies were
varied:
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Performing the mutagenesis
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References
Results — DNA Sequence Validation
Sequencing results of transformations that produced colonies were
varied:
Some colonies had no mutation.
Background
Performing the mutagenesis
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References
Results — DNA Sequence Validation
Sequencing results of transformations that produced colonies were
varied:
Some colonies had no mutation.
Others had multiple copies of the primer found at the point of
mutation.
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Performing the mutagenesis
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Results – Success!!
We obtained plasmids for three mutations.
Protein
FtsZ
ZipA
ZipA
Residue
Gly
Ala
Phe
Residue Number
21
193
Upstream in plasmid
References
Background
Performing the mutagenesis
Next steps
Final Remarks
Results – Success!!
We obtained plasmids for three mutations.
Protein
FtsZ
ZipA
ZipA
Residue
Gly
Ala
Phe
Residue Number
21
193
Upstream in plasmid
References
Background
Performing the mutagenesis
Contents
1
Background
2
Performing the mutagenesis
3
Next steps
4
Final Remarks
Next steps
Final Remarks
References
Background
Performing the mutagenesis
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Improving the yield
How do we avoid inserting multiple copies of the primer?
References
Background
Performing the mutagenesis
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Final Remarks
Improving the yield
How do we avoid inserting multiple copies of the primer?
One way is Single Primer Reactions IN Parallel (SPRINP),
proposed by Edelheit et al. [1].
Methylated DNA
plasmids
PCR
Forward primer
with mutation
Mix and run PCR cycles WITHOUT
using a polymerase
Methylated DNA
plasmids
PCR
Reverse primer
with mutation
References
Background
Performing the mutagenesis
“If I had more time . . . ”
Next steps
Final Remarks
References
Background
Performing the mutagenesis
“If I had more time . . . ”
SPRINP improvement.
Next steps
Final Remarks
References
Background
Performing the mutagenesis
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Final Remarks
“If I had more time . . . ”
SPRINP improvement.
Protein verification.
MALDI — amino acid sequence check.
Circular dichroism — secondary structure check.
Linear dichroism — mutated FtsZ polymerisation check.
References
Background
Performing the mutagenesis
Next steps
Final Remarks
“If I had more time . . . ”
SPRINP improvement.
Protein verification.
MALDI — amino acid sequence check.
Circular dichroism — secondary structure check.
Linear dichroism — mutated FtsZ polymerisation check.
Attach tags.
References
Background
Performing the mutagenesis
Next steps
Final Remarks
“If I had more time . . . ”
SPRINP improvement.
Protein verification.
MALDI — amino acid sequence check.
Circular dichroism — secondary structure check.
Linear dichroism — mutated FtsZ polymerisation check.
Attach tags.
Construct “artificial cells” containing tagged proteins
(SMALPs [5]).
References
Background
Performing the mutagenesis
Next steps
Final Remarks
“If I had more time . . . ”
SPRINP improvement.
Protein verification.
MALDI — amino acid sequence check.
Circular dichroism — secondary structure check.
Linear dichroism — mutated FtsZ polymerisation check.
Attach tags.
Construct “artificial cells” containing tagged proteins
(SMALPs [5]).
Observe using confocal microscopy.
References
Background
Performing the mutagenesis
“Artificial Cells” and SMALPs
Next steps
Final Remarks
References
Background
Performing the mutagenesis
Next steps
“Artificial Cells” and SMALPs
Membrane bound FtsZ — Osawa et al. [9].
Final Remarks
References
Background
Performing the mutagenesis
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Final Remarks
“Artificial Cells” and SMALPs
Membrane bound FtsZ — Osawa et al. [9].
Styrene Maleic Acid Lipid Particles (SMALP) — Knowles
et al. [5].
References
Background
Performing the mutagenesis
Contents
1
Background
2
Performing the mutagenesis
3
Next steps
4
Final Remarks
Next steps
Final Remarks
References
Background
Conclusions
Performing the mutagenesis
Next steps
Final Remarks
References
Background
Performing the mutagenesis
Next steps
Final Remarks
Conclusions
Site directed mutagenesis can be used as an alternative
approach to tagging proteins to study their kinetics . . . but
there are limitations.
References
Background
Performing the mutagenesis
Next steps
Final Remarks
Conclusions
Site directed mutagenesis can be used as an alternative
approach to tagging proteins to study their kinetics . . . but
there are limitations.
Protein verification – it remains to be seen whether the
mutated proteins expressed in this project retain key
properties of the wild-type form.
References
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Conclusions
Site directed mutagenesis can be used as an alternative
approach to tagging proteins to study their kinetics . . . but
there are limitations.
Protein verification – it remains to be seen whether the
mutated proteins expressed in this project retain key
properties of the wild-type form.
Beyond this, the next steps are to tag the proteins (the effect
of the tag will need to be considered), construct an artificial
cell containing these proteins and observe the resulting
interactions and Z-ring contraction.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
References
[1] Oded Edelheit, Aaron Hanukoglu, and Israel Hanukoglu. Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to
generate mutants for protein structure-function studies. BMC Biotechnology, 9(61):1–8, June 2009. ISSN 1472-6750. doi: 10.1186/1472-6750-9-61.
URL http://www.biomedcentral.com/1472-6750/9/61.
[2] Jean-Marc Ghigo, David S. Weiss, Joseph C. Chen, Justin C. Yarrow, and Jon Beckwith. Localization of FtsL to the Escherichia coli septal ring.
Molecular Microbiology, 31(2):725–737, 1999. ISSN 1365-2958. doi: 10.1046/j.1365-2958.1999.01213.x. URL
http://dx.doi.org/10.1046/j.1365-2958.1999.01213.x.
[3] Nathan W. Goehring and Jon Beckwith. Diverse Paths to Midcell: Assembly of the Bacterial Cell Division Machinery. Current Biology, 15(13):
R514–R526, July 2005. ISSN 0960-9822. doi: 10.1016/j.cub.2005.06.038. URL
http://linkinghub.elsevier.com/retrieve/pii/S0960982205006731.
[4] Younggyu Kim, Sam O. Ho, Natalie R. Gassman, You Korlann, Elizabeth V. Landorf, Frank R. Collart, and Shimon Weiss. Efficient Site-Specific
Labeling of Proteins via Cysteines. Bioconjugate Chemistry, 19(3):786–791, 2008. doi: 10.1021/bc7002499. URL
http://pubs.acs.org/doi/abs/10.1021/bc7002499. PMID: 18275130.
[5] Timothy J. Knowles, Rachael Finka, Corinne Smith, Yu-Pin Lin, Tim Dafforn, and Michael Overduin. Membrane Proteins Solubilized Intact in Lipid
Containing Nanoparticles Bounded by Styrene Maleic Acid Copolymer. Journal of the American Chemical Society, 131(22):7484–7485, 2009. doi:
10.1021/ja810046q. URL http://pubs.acs.org/doi/abs/10.1021/ja810046q.
[6] Anuradha Kuchibhatla, Anusri Bhattacharya, and Dulal Panda. ZipA Binds to FtsZ with High Affinity and Enhances the Stability of FtsZ
Protofilaments. PLoS ONE, 6(12):e28262, 12 2011. doi: 10.1371/journal.pone.0028262. URL
http://dx.doi.org/10.1371%2Fjournal.pone.0028262.
[7] Harry H. Low, Martin C. Moncrieffe, and Jan Löwe. The Crystal Structure of ZapA and its Modulation of FtsZ Polymerisation. Journal of Molecular
Biology, 341(3):839–852, 2004. ISSN 0022-2836. doi: 10.1016/j.jmb.2004.05.031. URL
http://www.sciencedirect.com/science/article/pii/S002228360400600X.
[8] William Margolin. FtsZ and the division of prokaryotic cells and organelles. Nature Reviews – Molecular Cell Biology, 6(11):862 – 871, November
2005. ISSN 1471-0072. doi: 10.1038/nrm1745. URL http://dx.doi.org/10.1038/nrm1745.
[9] Masaki Osawa, David E Anderson, and Harold P Erickson. Reconstitution of contractile FtsZ rings in liposomes. Science, 320(5877):792–794, May
2008. ISSN 1095-9203. doi: 10.1126/science.1154520. URL http://dx.doi.org/10.1126/science.1154520.
[10] Sambra D. Redick, Jesse Stricker, Gina Briscoe, and Harold P. Erickson. Mutants of FtsZ Targeting the Protofilament Interface: Effects on Cell
Division and GTPase Activity. Journal of Bacteriology, 187(8):2727–2736, 2005. doi: 10.1128/JB.187.8.2727-2736.2005. URL
http://jb.asm.org/content/187/8/2727.abstract.
[11] Elaine Small, Rachel Marrington, Alison Rodger, David J. Scott, Katherine Sloan, David Roper, Timothy R. Dafforn, and Stephen G. Addinall. FtsZ
Polymer-bundling by the Escherichia coli ZapA Orthologue, YgfE, Involves a Conformational Change in bound GTP. Journal of Molecular Biology, 369
(1):210 – 221, 2007. ISSN 0022-2836. doi: 10.1016/j.jmb.2007.03.025. URL
http://www.sciencedirect.com/science/article/pii/S002228360700366X.
[12] Miguel Vicente and Jan Lowe. Ring, helix, sphere and cylinder: the basic geometry of prokaryotic cell division. EMBO Reports, 4(7):655–660, 2003.
ISSN 1469-221X. doi: 10.1038/sj.embor.embor885. URL http://dx.doi.org/10.1038/sj.embor.embor885.
Background
Performing the mutagenesis
Next steps
Final Remarks
Acknowledgements
Claire Dow & David Roper
All of the Structural Biology Lab (C10) at Gibbet Hill
MOAC DTC staff and fellow students
The University of Warwick, EPSRC and BBSRC
References
Background
Performing the mutagenesis
Next steps
Final Remarks
Acknowledgements
Claire Dow & David Roper
All of the Structural Biology Lab (C10) at Gibbet Hill
MOAC DTC staff and fellow students
The University of Warwick, EPSRC and BBSRC
Thank you for listening! :-) Any questions?
References