SPARQED IMMERSION PROGRAM 27 th of June 1

SPARQ-ED IMMERSION PROGRAM 27 th of June – 1 st of July Name. Research 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. Name. Research 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. Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

Gap 2 (G 2)- Cell prepares to divide. Mitosis (M)- Cell divide occurs. Gap 1 (G 1)- The cell grows and prepares to synthesize. DNA Synthesis (S-Phase) - The cell synthesizes- the replication of the DNA strands. (THE MOST KEY STAGE) Where h. SSB 1 comes into place. Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

INTRODUCTION Slide sub-heading (manual text box) 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. Name. Research 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 h. SSB 1), repairs damaged DNA, inactivating/intercepting mutagenic molecules Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

INTRODUCTION PROTEINS What is a protein? A group of amino acids joined together by peptide bonds. Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

INTRODUCTION PROTEINS Functions of proteins, • Structural support – collagen • Defensive – antibodies • Storage – ferritin • DNA damage signaling – h. SSB 1 The protein that we are looking at during this immersion program is h. SSB 1 Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

INTRODUCTION h. SSB 1 Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

RESEARCH QUESTION What Is the effect of h. SSB 1 mutations on the repair process of DNA damage? Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

Project Overview Transformation Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

DAY 1 METHOD • • Transformation of cells Construct and culture bacteria into larger populations Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

DAY 1 METHOD MUTANTS: -2 Point Mutations -2 Point Truncations -2 Wild Types Name. Research 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 Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

Day 1: Results Controlled Unsuccessful Transformation Name. Research 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 Name. Research 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 Name. Research 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 (1 L) , 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 Name. Research 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 5000 rpm 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 20 ml 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 50 ml centrifuge tube, so it could be further refined, and the supernatant was again removed (4000 rpm for 15 minutes). The pellet was then frozen at -80° Celcius overnight Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

DAY 2 RESULTS Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

DAY 2 RESULTS Name. Research 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 Name. Research 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 Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

DAY 3 & 4 METHOD Sonication Washes and Elution Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

DAY 3 & 4 METHOD Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

DAY 3 & 4 WASHES Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

DAY 3 & 4 ELUTIONS Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

DAY 3 & 4 ELUTIONS Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

DAY 3 & 4 RESULTS and DISCUSSION Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

DAY 3 & 4 EMSA Used to determine which proteins will bind to DNA Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

DAY 3 & 4 EXAMPLES OF EMSA GELS Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

CONCLUSION RESULTS The experimental hypothesis “If you mutate h. SSB 1 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 • h. SSB 1 can be replicated and purified in laboratory conditions to test its effect on DNA repair. Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

CONCLUSION h. SSB 1 • Essential Single Stranded Binding protein • Vital role in recruiting the MRN complex and ATM to SSBs • Maintains genomic stability • Measure of aggressiveness of cancer Name. Research 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. Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

FUTURE IMPLICATIONS Hallmarks of Cancer that h. SSB 1 prevents Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

FUTURE IMPLICATIONS Future Application of Mutations of h. SSB 1 Protein Mutations of h. SSB 1 protein could be used to kill cancer cells • h. SSB 1 has never been found naturally mutated Research possible drug that can deactivate h. SSB 1 amino acid in cancer cells • Time to develop drug • What can deactivate h. SSB 1 amino acids? Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

Mutation of TRP 55 to ALA disconnects h. SSB 1 from the DNA How can other mutations can affect the structure of the protein? Figure 1: TRP 55 without Mutation Figure 2: (RED) ALA Mutation (RED) Name. Research 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 h. SSB 1 acts in the early stages of DNA damage response Research the interactions of h. SSB 1 with MRN when DNA damage response is initiated Name. Research 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 Mc. Millan Mark Adams Joe Groth Kath Hampson Lions Medical Research Foundation Name. Research of presentation Month 2008 SPARQ-ed Immersion Project

Thank You Everyone! Name. Research 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 Name. Research of presentation Month 2008 SPARQ-ed Immersion Project