SPARQ-ED IMMERSION PROGRAM

1. SPARQ-ED IMMERSION PROGRAM 27th of June – 1st of July

2. 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.

3. INTRODUCTIONTHE 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.

4. Mitosis (M)- Cell divide occurs. Gap 2 (G2)- Cell prepares to divide. Gap 1 (G1)- 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 hSSB1 comes into place.

5. INTRODUCTIONCANCER • Slide sub-heading (manual text box) • CANCER AND ITS CAUSE • Cancer - class of diseases characterized by the uncontrolled division of abnormal cells in a part of the body • Cancer caused by acquired mutations • Causes of mutations – dividing cells, hereditary or epigenetic (e.g. chemicals from tobacco smoke) factors • Mutations prevent cells from functioning normally (e.g. providing too much protein) • Cells are more likely to develop further mutations and less likely to be able to repair the damaged genes if they are abnormal. • Around half a dozen mutations turns a normal cell into a cancer cell.

6. INTRODUCTIONCANCER • Slide sub-heading (manual text box) • HALLMARKS OF CANCER • Hallmarks of cancer - sustaining proliferate signals, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis • Genome instability - increased rates of mutations in order to accumulate several mutations needed to foster tumorigenesis • Cancer cells alter DNA-maintenance machinery (caretaker genes) • Roles of ‘caretaker’ genes - detects DNA damage and activates repair machinery (e.g hSSB1), repairs damaged DNA, inactivating/intercepting mutagenic molecules

7. INTRODUCTIONPROTEINS • What is a protein? • A group of amino acids joined together by peptide bonds.

8. INTRODUCTIONPROTEINS • 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

9. hSSB1 INTRODUCTION hSSB1

10. RESEARCH QUESTION • What Is the effect of hSSB1 mutations on the repair process of DNA damage?

11. Project Overview Transformation

12. DAY 1METHOD • Transformation of cells • Construct and culture bacteria into larger populations

13. DAY 1METHOD • MUTANTS: • -2 Point Mutations • -2 Point Truncations • -2 Wild Types

14. DAY 1RESULTS • 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

15. Day 1: Results Controlled Unsuccessful Transformation

16. DAY 1DISCUSSION • 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

17. DAY 1DISCUSSION 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

18. DAY 2METHOD 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

19. 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

20. DAY 2RESULTS

21. DAY 2 • RESULTS

22. DAY 2DISCUSSION • LARGE CULTURE GROWTH • Begun with small culture of bacteria, resulted in much larger pellet of bacteria cells • Large pellet indicates that bacterial replication has been successful • IPTG INTRODUCTION • Process of induction is most efficient at the peak of bacteria replication • For the most efficient induction, IPTG was added between 0.6 and 0.8 optical density (OD) Live bacteria

23. DAY 2DISCUSSION • 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

24. DAY 3 & 4METHOD • Sonication Washes and Elution

25. DAY 3 & 4METHOD

26. DAY 3 & 4WASHES

27. DAY 3 & 4ELUTIONS

28. DAY 3 & 4ELUTIONS

29. DAY 3 & 4RESULTS and DISCUSSION

30. DAY 3 & 4EMSA • Used to determine which proteins will bind to DNA

31. DAY 3 & 4EXAMPLES OF EMSA GELS

32. 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.

33. 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

34. 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.

35. FUTURE IMPLICATIONS • Hallmarks of Cancer that hSSB1 prevents

36. FUTURE IMPLICATIONS • Future Application of Mutations of hSSB1 Protein

37. 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 (RED) Figure 2: ALA Mutation (RED)

38. FUTURE IMPLICATIONS • Development of Anti-Cancer Drugs

39. 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

40. Thank You Everyone!

41. Thank You Everyone!Please head outside and stay to socialise and for afternoon tea! • Wei-Han Chan • Jack Armstrong • Emma Simpson • Holly Wilson • Jade Wilson • Emma Hansen • Shambhavi Srivastava • Emma Sleight • Sophie Taylor • Montana-Adelen Olm • Trinity Wong • AraniyaMaharaj • Jazmyn Johansen • Gayathri Nair • Abigail White • Ainsley Robertson • SuresanArraviind • HeilynBonquin • Luke Liu • Ally Chen • BreannanBusetti