After choosing appropriate restriction enzymes, the researcher digests both the plasmid and the insert with the selected restriction enzymes in separate 50µl reactions with a final DNA concentration (plasmid or insert, respectively) of 10 ng/µL.

Q28. Based on the amount of insert DNA calculated to achieve a 3:1 ratio of insert to plasmid, calculate the volume of digested insert DNA that the researcher must add to the ligation reaction. Record your answer to two decimal places and show your calculations.

Q23. Use the experimental data provided throughout this case study to explain the molecular and cellular mechanisms contributing to the development of colorectal cancer in this patient. In your answer, link the APC sequence variant to downstream effects on Wnt signalling and explain how altered apoptosis regulation contributes to tumour formation. (10 marks)

Figure 6. Western blot detection of Bak, Bax and Bcl-2 proteins in patient-derived colorectal cancer intestinal epithelial cells (IECs) and normal IECs.

Apoptosis was then assessed functionally following camptothecin treatment by examining DNA fragmentation using agarose gel electrophoresis and executioner caspase activation assays. The executioner caspase activation assay demonstrated robust executioner caspase activation in the normal IECs following treatment, whereas no executioner caspase activation was detected in the patient-derived colorectal cancer IECs.

Figure 7. Three potential agarose gel electrophoresis results (A-C) for the DNA fragmentation assay. M = DNA ladder, 1 = normal IECs without camptothecin treatment, 2 = normal IECs following camptothecin treatment, 3 = colorectal cancer IECs following camptothecin treatment.

One hallmark of cancer cells is their ability to resist programmed cell death. To investigate whether the patient’s colorectal tumour cells are defective in their ability to undergo apoptosis, the researcher performed a series of apoptosis assays. Patient-derived colorectal cancer intestinal epithelial cells (IECs) and normal IECs were treated with the chemotherapeutic agent camptothecin which is a topoisomerase inhibitor.

To investigate the molecular regulation of intrinsic apoptosis, total protein was extracted from the patient-derived IECs and normal IECs, and western blotting was performed to detect key regulators of intrinsic apoptosis including Bak, Bax and Bcl-2. Bak, Bax and Bcl-2 were detected using HRP-conjugated antibodies and Clarity ECL Western Blotting Substrate from Bio-Rad. The western blotting results are shown in Figure 6. 

Q22. Predict whether the patient’s colorectal cancer cells are likely to respond to treatment with a BH3 mimetic. Use the data provided in this case study to justify your prediction. (2-3 sentences) (2 marks)

Q19. Clarity ECL Western Blotting Substrate is classified as a hazardous chemical. Use Chemwatch to locate the miniSDS for Clarity ECL Western Blotting Substrate (CW No. 900-2477) and upload a copy to the file upload box below.

The colorectal tumour tissue and normal colon tissues resected from the patient were dissociated into single cell suspensions. Intestinal epithelial cells (IECs) were isolated from the suspensions and cultured in vitro. The IECs were labelled with a GFP-tagged anti-β-catenin antibody and DAPI. The results are shown in Figure 5.

Figure 5. Fluorescent microscopy images showing β-catenin localisation in intestinal epithelial cells (IECs) isolated from patient-derived colorectal tumour tissue and normal colon tissue.