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SPARQ-ED
IMMERSION
PROGRAM
27th of June – 1st of July
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
WELCOME TO COUNTRY
I would like to respectfully acknowledge the Turrbal and
Jagera People, the Traditional Owners of the land on which
this event is taking place and Elders both past and present.
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
INTRODUCTION
THE CELL CYCLE
•
Cell cycle is the four stages that occur during the division of a cell.
•
The most critical of the four is the replication of the DNA strands.
•
In a normal cell it’s controlled by a complex series of signaling
pathways.
•
Has a wide variety of mechanisms to ensure that most errors in cells
are corrected if not apoptosis occurs.
•
Apoptosis- The death of a cell without releasing harmful substances
to the surrounding area. The elimination of old, unnecessary or
unhealthy cells.
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
Gap 2 (G2)- Cell
prepares to divide.
Mitosis (M)- Cell
divide occurs.
Gap 1 (G1)- The cell
grows and prepares
to synthesize.
DNA Synthesis (SPhase)- The cell
synthesizes- the
replication of the DNA
strands. (THE MOST
KEY STAGE)
Where hSSB1 comes
into place.
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
INTRODUCTION
Slide sub-heading (manual text box)
CANCER
CANCER AND ITS CAUSE
o
Cancer - class of diseases characterized by the uncontrolled division
of abnormal cells in a part of the body
o
Cancer caused by acquired mutations
o
Causes of mutations – dividing cells, hereditary or epigenetic (e.g.
chemicals from tobacco smoke) factors
o
Mutations prevent cells from functioning normally (e.g. providing too
much protein)
o
Cells are more likely to develop further mutations and less likely to
be able to repair the damaged genes if they are abnormal.
o
Around half a dozen mutations turns a normal cell into a cancer cell.
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
INTRODUCTION
Slide sub-heading (manual text box)
CANCER
HALLMARKS OF CANCER
o
Hallmarks of cancer - sustaining proliferate signals, evading growth
suppressors, resisting cell death, enabling replicative immortality,
inducing angiogenesis, activating invasion and metastasis
o
Genome instability - increased rates of mutations in order to
accumulate several mutations needed to foster tumorigenesis
o
Cancer cells alter DNA-maintenance machinery (caretaker genes)
o
Roles of ‘caretaker’ genes - detects DNA damage and activates
repair machinery (e.g hSSB1), repairs damaged DNA,
inactivating/intercepting mutagenic molecules
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
INTRODUCTION
PROTEINS
What is a protein?
A group of amino acids joined together by peptide bonds.
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
INTRODUCTION
PROTEINS
Functions of proteins,
•
Structural support – collagen
•
Defensive – antibodies
•
Storage – ferritin
•
DNA damage signaling – hSSB1
The protein that we are looking at during this immersion program is hSSB1
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
INTRODUCTION
hSSB1
hSSB1
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
RESEARCH QUESTION
What Is the effect of
hSSB1 mutations on the
repair process of DNA
damage?
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
Project Overview
Transformation
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
DAY 1
METHOD
•
•
Transformation of cells
Construct and culture bacteria into larger populations
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
DAY 1
METHOD
MUTANTS:
-2 Point Mutations
-2 Point Truncations
-2 Wild Types
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
DAY 1
RESULTS
Transformation: The genetic alteration of a cell resulting from the direct uptake and incorporation
of exogenous DNA through the cell membrane
E.coli isolated by kanamycin
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
Day 1: Results
Controlled
Unsuccessful Transformation
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
DAY 1
DISCUSSION
The successful growth of bacterial colonies shows that the
transformation of the bacterial cells was successful, while any
colonies that did not develop showed an unsuccessful
transformation.
The successful transformations enabled growth because:
• The agar plates contained the antibiotic Kanamycin, which
kills any cells that do not contain the resistance gene.
• Only cells that transformed contained the resistance gene,
which would enable them to form colonies
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
DAY 1
DISCUSSION
If transformation was unsuccessful, the bacteria cells would
not develop because:
• They had not taken in the plasmid containing the
Kanamycin resistance gene
• They would die on the agar plates, because they were
not resistant to the Kanamycin antibiotic
The transformation process may not have been successful
because:
• Competent cells may not have been effective
• Heat shock process may not have been correctly
conducted, e.g. kept at 42° for too long or not long
enough
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
DAY 2
METHOD
Large Scale Bacterial Culture and Protein Induction
The larger scale bacterial culture was performed by
pipetting the culture overnight inside a large flask of LB
Broth (1L) , and this was done as the LB Broth is
essentially food for the bacteria, so they can replicate.
Figure 1 –
Colony Picking
Figure 2 Sampling
Figure 3 Spectrometry
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
Method (Cont.)
The flask was then emptied into a container
so it could be centrifuged. It was centrifuged
so a pellet forms, to collect the bacteria. The
centrifuge was performed at 5000rpm for 10
mins and then produced a pellet as pictured
(right).
Following the centrifuge, the supernatant (the
liquid above the pellet) was all removed apart
from 20ml to make sure the pellet doesn't dry
out.
The pellet was then resuspended in the
solution.
After the resuspension, it was poured into a
50ml centrifuge tube, so it could be further
refined, and the supernatant was again
removed (4000rpm for 15 minutes). The
pellet was then frozen at -80° Celcius
overnight
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
DAY 2
RESULTS
NameResearch
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Month
2008
SPARQ-ed
Immersion
Project
DAY 2
RESULTS
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
DAY 2
DISCUSSION
LARGE CULTURE GROWTH
IPTG INTRODUCTION
•Begun with small culture of bacteria,
resulted in much larger pellet of
bacteria cells
•Process of induction is most
efficient at the peak of bacteria
replication
•Large pellet indicates that bacterial
replication has been successful
•For the most efficient induction,
IPTG was added between 0.6 and
0.8 optical density (OD)
Live bacteria
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
DAY 2
DISCUSSION
ELECTROPHORESIS GEL
•
How do we know which is our protein?
•
The overexpressed component which is not present in the
control is generally our specific protein.
•
Some proteins are too small for the pores; resulting in them
flowing through the gel to the bottom.
C
1
2
3
C
4
5
6
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
DAY 3 & 4
METHOD
Sonication
Washes and Elution
NameResearch
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Month
2008
SPARQ-ed
Immersion
Project
DAY 3 & 4
METHOD
NameResearch
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Month
2008
SPARQ-ed
Immersion
Project
DAY 3 & 4
WASHES
NameResearch
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Month
2008
SPARQ-ed
Immersion
Project
DAY 3 & 4
ELUTIONS
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
DAY 3 & 4
ELUTIONS
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
DAY 3 & 4
RESULTS and DISCUSSION
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
DAY 3 & 4
EMSA
Used to determine which proteins will bind to
DNA
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
DAY 3 & 4
EXAMPLES OF EMSA GELS
NameResearch
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Month
2008
SPARQ-ed
Immersion
Project
CONCLUSION
RESULTS
The experimental hypothesis “If you mutate hSSB1 it will
have an effect on the DNA repair process” was supported.
From these results it was seen that:
• Mutations can be induced
• Protein can be isolated
• hSSB1 can be replicated and purified in laboratory
conditions to test its effect on DNA repair.
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
CONCLUSION
hSSB1
• Essential Single Stranded
Binding protein
• Vital role in recruiting the MRN
complex and ATM to SSBs
• Maintains genomic stability
• Measure of aggressiveness of
cancer
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
CONCLUSION
CELL CYCLE AND CANCER
•
Cell cycle is composed of four
phases.
•
Cancer arises when breakdown of
regulatory roles of checkpoints
allows cells with errors to enter
mitosis and hence pass it on to
daughter cells.
•
If there is an interference with cell
cycle, cells can enter into state of
continuous division, a hallmark of
cancer.
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
FUTURE IMPLICATIONS
Hallmarks of Cancer that hSSB1 prevents
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
FUTURE IMPLICATIONS
Future Application of Mutations of hSSB1 Protein
Mutations of hSSB1 protein could be
used to kill cancer cells
•hSSB1 has never been found naturally mutated
Research possible drug that can deactivate
hSSB1 amino acid in cancer cells
• Time to develop drug
• What can deactivate hSSB1 amino acids?
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
Mutation of TRP55 to ALA disconnects
hSSB1 from the DNA
How can other mutations can affect the structure of the protein?
Figure 1:
TRP55 without Mutation
Figure 2:
(RED)
ALA Mutation
(RED)
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
FUTURE IMPLICATIONS
Development of Anti-Cancer Drugs
Focus on inhibition of DNA repair process disrupting the
Hallmarks of Cancer
Drug development can be aided by investigating how
hSSB1 acts in the early stages of DNA damage response
Research the interactions of hSSB1 with MRN when DNA
damage response is initiated
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
THANK YOU TO:
Associate Professor Derek Richard
Anne Brant
Shannon Walsh
Syed Ali Naqi Raza Jaffray
Mark Fisher
Marcos Riba
Fiona McMillan
Mark Adams
Joe Groth
Kath Hampson
Lions Medical Research Foundation
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
Thank You Everyone!
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project
Thank You Everyone!
Please head outside and stay to socialise and for
afternoon tea!
Wei-Han Chan
Jazmyn Johansen
Jack Armstrong
Gayathri Nair
Emma Simpson
Abigail White
Holly Wilson
Ainsley Robertson
Jade Wilson
Suresan Arraviind
Emma Hansen
Heilyn Bonquin
Shambhavi Srivastava
Luke Liu
Emma Sleight
Ally Chen
Sophie Taylor
Breannan Busetti
Montana-Adelen Olm
Trinity Wong
Araniya Maharaj
NameResearch
of presentation
Month
2008
SPARQ-ed
Immersion
Project