BMB Course Handbook 2018-19 PDF

Biochemistry & Molecular Biology Department of Biochemistry Natural Sciences Tripos Part IB Biochemistry & Molecular Biology ATP Synthase Course Handbook 2018-19 Biochemistry & Molecular Biology (BMB) NST 2018-19 Biochemistry & Molecular Biology Course Handbook 2018-19 Table of Contents Aims and Objectives for Biochemistry & Molecular Biology 9 Introduction to Biochemistry & Molecular Biology 10 BMB Moodle Site 10 Lectures 12 Lecture Recordings – Panopto 12 Practical Classes 13 Journal Clubs 17 Experimental Design Exercise 17 Examinations 17 BMB Lecture Timetable 2018-19 19 BMB Practical Timetable 2018-19 21 Reading List 23 Recommended books 23 Other sources of information 25 Plagiarism 26 Synopsis of the BMB lecture course 29 Michaelmas Term: Genes and proteins – macromolecules in action 29 Lent Term: Energy transduction, cell signalling and cell proliferation 30 Easter Term: Biochemistry of Microorganisms 32 Course Management and Student Feedback 34 Safety in the Biochemistry Part I Practical Teaching Laboratory 37 Safety Acknowledgement and Compliance Form 40 Health Record Form – Record of Hazardous Substance Usage 41 Risk Assessments 42 Appendix 1: Units – prefixes, amount and concentration 51 Units 51 Prefixes 51 The distinction between amount and concentration 51 Amount 51 Concentration 52 Dilutions 52 Appendix 2: Acids, Bases, pH and Buffers 53 Why are acid-base concepts important in Biochemistry? 53 What are protons, acids and bases? 53 Logarithms: definition of pH 54 The Henderson-Hasselbalch equation and its uses 54 1 © Department of Biochemistry, 2018 Biochemistry & Molecular Biology (BMB) NST 2018-19 Properties of buffers 57 Types of buffer useful for experimental purposes 58 Some Exercises 59 Answers to exercises 60 Appendix 3: Principles of Spectrophotometry 61 Absorption of light 61 Some practical points about measuring absorbance 63 Weeks 1-4 (Michaelmas) – Basic Techniques in Molecular Biology 65 Summary of Schedule 66 Week 1 – Introduction and background 67 Week 1: PCR and Cloning – Experimental 73 Week 2 – Introduction and background 81 Week 2 – Basic Techniques in Biochemistry – Experimental 85 Week 3 – Introduction and background 93 Week 3 – Protein Purification – Experimental 95 Week 4 – Introduction and background 103 Week 4 – Electrophoresis Mobility Shift Assay – Experimental 105 Question Sheets – Practicals 1 to 4 109 Week 1 – Questions 109 Week 2 – Questions 113 Week 3 – Questions 114 Week 4 – Questions 115 Week 5: BMB Experimental Exercise – Using Online Databases as Tools 117 Summary of what you will achieve in this exercise – you will aim to: 118 1. Investigating the origin of the peptides 118 2. Finding the nucleotide sequence encoding your protein 123 3. Identifying the Open Reading Frame (ORF) 124 4. Identifying the cDNA sequence corresponding to the ORF 125 5. Cloning the cDNA encoding the ORF into a suitable expression vector 126 6. Designing Primers 127 7. RT-PCR 131 8. RNAi to knockdown your protein of interest 131 Other databases 134 Glossary 135 Week 6: Discussion of Practicals from Weeks 1–5 139 Week 7 Practical 140 Week 8 – Analysis of Protein Structure by Molecular Graphics 141 Objectives 141 Introduction 141 Section 1: Analysis of the covalent connectivity of the polypeptide chain 143 2 © Department of Biochemistry, 2018 Biochemistry & Molecular Biology (BMB) NST 2018-19 Section 2: Analysis of protein secondary structure – alpha helices 147 Section 3: Analysis of protein secondary structure – beta strands 151 Section 4: Super-secondary structure – recurrent structural motifs in globular proteins. 152 Section 5: Functional motifs 154 Section 6: Analysis of the tertiary structure of proteins 155 Section 7: Analysis of the structural basis of protein function 158 Section 8: Some interesting structures 162 Appendix 4: The 20 common amino acids: 165 Week 1 (Lent) – Subcellular Fractionation of Mammalian Tissues 169 Objectives 169 Background 169 Subcellular Fractionation – Experimental 173 Subcellular Fractionation – Results and Assessment 183 Week 2 (Lent) Portrait of an Enzyme: Chymotrypsin – 185 Objectives 185 Simple Kinetics 188 Methods for estimating Km and Vmax 191 Irreversible inhibitors 194 pH Dependence of Chymotrypsin Catalysis 195 Enzyme Kinetics – Experimental 199 Enzyme Kinetics – Use of the Computer 201 Enzyme Kinetics – Question and Assessment Sheet 203 Week 3: Metabolic Control Analysis Practical 211 Metabolic Control Analysis in a Nutshell: 211 Getting to Know the Kinetic Model Used in the Practical: 212 COPASI as a Tool for Building and Investigating Kinetic Models: 215 Getting to know the model: (20 min) 217 Getting ready to run simulations: (10 min) 218 Running simulations using the model: (15 min) 220 The effect of PFK concentration per unit time on the glycolytic flux – Determination of a flux control coefficient at steady state: (40 min) 220 The effect of TPI enzyme activity on the glycolytic flux – Determination of a Flux Control Coefficient at steady state: (25 min) 221 F26bP as a regulator of the glycolytic flux: (35 min) 222 Metabolic Control Analysis – Questions Addressed in the Practical 225 Week 4 (Lent) – Mitochondrial Oxidative Phosphorylation 227 Objectives 227 Introduction and Background 227 Oxidative Phosphorylation – Experimental 229 A. Rates of respiration and P:O ratios with succinate 229 3 © Department of Biochemistry, 2018 Biochemistry & Molecular Biology (BMB) NST 2018-19 B. Substrates and inhibitors of the mitochondrial respiratory chain 230 C. Control of respiration by ATP production and consumption 231 D. Design an experiment to test how a toxin works on mitochondria 232 Oxidative Phosphorylation – Results 235 A. Rates of respiration and P:O ratio with succinate 235 B. Substrates and inhibitors of the mitochondrial respiratory chain 235 C. Control of respiration by ATP production and consumption 236 D. Design an experiment to test how a toxin works on mitochondria 237 Cell Signalling 1. Tyrosine Phosphorylation in Platelets 239 Objectives 239 Introduction 239 Summary of procedure: Week 1 241 Summary of procedure: Week 2 241 Some background about antibodies 242 Cell Signalling I – Experimental procedure – Week 1 245 1. Prepare the polyacrylamide gel 245 2. Platelet assays 246 3. Gel loading 247 4. Setting up the Western blot 247 Cell Signalling I – Experimental procedure – Week 2 248 Ponceau staining 248 Probing with Antibodies 248 Detection 248 Cell Signalling I – Question Sheet 251 Cell Signalling 2: The production of Thromboxane by human platelets 253 Objectives 253 Introduction and Background 253 Week 7 – Cell Signaling: The production of Thromboxane by Human Platelets – Experimental 259 Cell Signalling 2: Experimental Procedure 260 Data Handling and Interpretation – Week 2 265 Drawing the standard curve 265 Determining the amount of TxB2 in your samples 266 Dose response curve 266 Converting the concentrations to biologically meaningful values 266 Cell Signalling 2 – Results and Questions 269 Protein targeting and “global” gene regulation in bacteria. 273 Background to the practical 273 A. Protein targeting and use of TnblaM positive selection. 273 B. “Global” regulatory mutations in bacteria. 274 C. Environmental bacteria that make signalling molecules or antibiotics. 274 4 © Department of Biochemistry, 2018 Biochemistry & Molecular Biology (BMB) NST 2018-19 Day 1 – Experimental Procedure 275 Note: Safety and aseptic technique 275 A. Secondary transposition, “high hopping” and tagging targeted proteins 275 B. Mutants, pleiotropy and “global” gene regulation in bacteria 276 C. Environmental bacteria that make signalling molecules or antibiotics. 276 Day 2 – Experimental Procedure 277 A. Secondary transposition, “high hopping” and tagging targeted proteins. 277 B. Mutants, pleiotropy and “global” gene regulation in bacteria 277 C. Environmental bacteria that make signalling molecules or antibiotics. 277 Day 2 – Questions: 281 5 © Department of Biochemistry, 2018 Biochemistry & Molecular Biology (BMB) NST 2018-19 6 © Department of Biochemistry, 2018 University of Cambridge Department of Biochemistry NATURAL SCIENCES TRIPOS PART IB Biochemistry & Molecular Biology (BMB) Course Handbook Course Organiser: Dr Dee Scadden (Email: [email protected]) Biochemistry Sanger Building: Biochemistry Sanger Building: Lectures Practicals 7 © Department of Biochemistry, 2018 The Department of Biochemistry recognises that each student has their own optimal way of studying and learning. Please take time to read this course handbook to see what different resources are available to you, and to try them out to see what works best for you. 8 © Department of Biochemistry, 2018 BMB Aims and Objectives NST 2018-19 Aims and Objectives for Biochemistry & Molecular Biology Aims The course aims to build on the NST IA Biology of Cells course, and to provide an advanced foundation for specialist further study of Biochemistry or other molecular biosciences, by giving students an understanding of: 1) the structural organisation of genes and the control of gene expression in prokaryotes and eukaryotes; 2) protein structure, enzyme catalysis and protein engineering; 3) the control of metabolic pathways, energy transduction and cell growth; 4) the methods used to analyse biochemical structures and processes, including the use of molecular genetic tools. Objectives By the end of the course, the students should be able to demonstrate knowledge and understanding of: 1) The principles and exploitation of methods of recombinant DNA technology 2) Chromatin structure, RNA synthesis, processing and translation 3) Protein domain structure and folding, conformational mobility and stability, enzyme kinetics, enzyme mechanisms, allostery and antibody recognition and protein design 4) The structural basis and mechanism of energy transduction processes in mitochondria, photosynthetic bacteria and chloroplasts, and of the control of metabolic flux 5) The control of eukaryotic cell cycle 6) Signal transduction across membranes and within and between cells 7) Bacterial chemotaxis, motility and secretion systems 8) Aspects of the molecular biology of protozoa: evolution, disease causation, antigenic variation, and regulation of gene expression in trypanosomes and be skilled in: 9) The design and execution of simple experimental protocols, be skilled in the use of laboratory equipment to obtain reproducible data, and to be able to use computer applications to analyse gene and protein sequences and structures 10) Analysis and critical interpretation of the results of biochemical experiments, using examples from their own laboratory practice, journal clubs and lectures and continue their learning by: 11) Building on their knowledge, understanding and skills by further study of specialised biomolecular courses within the Natural Sciences Tripos. 9 © Department of Biochemistry, 2018 BMB Introduction NST 2018-19 Introduction to Biochemistry & Molecular Biology BMB Moodle Site We provide a website for the Biochemistry & Molecular Biology (BMB) course using the University’s Virtual Learning Environment (VLE), Moodle, accessed from www.vle.cam.ac.uk. You will be registered for the BMB site once we have collected your User IDs at the practical registration. There is a lot of important information on Moodle: Look out for course announcements Information about the course is provided on Moodle Moodle provides on-line access to course materials and resources: E.g. lecture handouts, films of lectures (via Panopto – see below), pre-practical quizzes, interactive practical materials, exam quizzes, background information (E.g. techniques posters and films), information for the practical classes, tutorial films, etc. Additional material to support the BMB course will be provided on Moodle throughout the academic year – do keep an eye out for new additions or developments. Do take the time to explore the BMB Moodle site, as there are many resources that will support your learning, both for lectures and practical classes. Please note that there are resources on Moodle that are absolutely essential for your course work. In addition, there are also a large number of additional resources that have been provided to support your learning (E.g. quizzes, interactive practical materials, extra experimental design exercises, information about biochemical techniques etc.), and it is not essential that you complete or engage with all of the material provided. The virtual learning environment should support the way YOU learn best, so feel free to pick and choose from the resources provided to use those best suited to your individual learning style. A few practical details about using Moodle for BMB The format of the BMB Moodle site may be arranged slightly different to what you’ve used previously. Here are a few helpful tips to get you started: 10 © Department of Biochemistry, 2018 BMB Introduction NST 2018-19 Helpful information can be found in the blocks on the right-hand side of Moodle Click on each coloured box on the front page to access the various course materials or information Lecture materials etc. in each block are generally organised into ‘Moodle Books’, where there are separate Moodle Books for each lecture set. E.g. Within the block ‘BMB Lectures – Michaelmas Term’: To access the contents of Moodle Books, click on the book and the chapters will appear in the top right-hand side of the screen, and the contents of each chapter will be in the middle of the screen: E.g. Chapter 1: Lecture handout 2017 All of the materials on the BMB Moodle site can be accessed by o Navigating from the front page (click on the top image (collage) at any time to take you back to the BMB front page) o Using the drop-down menu in each section ‘Jump to…’ o Using the navigation arrows/text at the sides of each section E.g. Some course information will also be available via the Biochemistry Department at http://www.bioc.cam.ac.uk/teaching/second-year/bmb 11 © Department of Biochemistry, 2018 BMB Introduction NST 2018-19 Lectures Lecture handouts are provided in advance on Moodle and in hard copy at the lectures. To aid discussion in supervisions, lecturers will provide some questions related to the lecture material, if you and your supervisors wish to use them. Your supervisors will be provided with guides to answering these questions. Various resources to support the lectures are included on Moodle. These are as follows: Lecture handouts Lecture slides Questions based on the lectures as provided by lecturers Literature sources, reading lists etc. Lecture recordings – via Panopto software (see below) Lecture Recordings – Panopto The majority of lectures in BMB will be recorded using ‘Panopto’ and will be accessible via the BMB Moodle site. Here are a few tips for using Panopto for BMB. Lecture recordings for BMB can be accessed in two ways: o via the Panopto block (top right-hand side of BMB Moodle page) where the recordings will be listed by date o via links in the Moodle book for each set of lectures 12 © Department of Biochemistry, 2018 BMB Introduction NST 2018-19 You can search for key words in recordings – these will be identified by voice recognition and from text on slides: E.g. if you type ‘translation’ in search box (top left-hand side of Panopto screen), you will get a list of places where that word occurs, and at what time. You can then skip forward to the part you are looking for. You can also use bookmarks to mark bits that you might want to go back to later: Practical Classes Aim of the practicals Progress in science is achieved through observation and experiment. Biochemistry (and its close cousin, molecular biology) is an experimental science that advances from well-thought out investigations in the laboratory. No serious student should neglect the opportunities that this course provides to appreciate this fact. Your course includes experiments devised for you to gain some insight into how laboratory investigations are carried out and how data are processed and interpreted. To obtain useful results an experiment should be designed to answer a definite question and the detailed planning should be sufficiently rigorous to exclude adventitious errors. The course gives you the opportunity to plan some experiments for yourselves. You should benefit from the practicals in three ways: (i) You will learn a variety of experimental techniques, all of which are currently used in biochemical research. The practicals have been designed to complement the lectures and fit in with their sequence as far as possible. The hands-on experience should link 13 © Department of Biochemistry, 2018 BMB Introduction NST 2018-19 to the mental framework provided by the lectures, and give you a deeper understanding and more realistic perspective of the topics discussed. (ii) You will learn to handle experimental data effectively, and to extract the maximum information content without falling into the trap of over-interpretation. (iii) You will be helped when it comes to the data handling questions in the Tripos examination. A collection of question papers from previous years is available on the course website. Quizzes based on the previous papers can also be found on Moodle. The Demonstrators Demonstrators are there to help you. The senior demonstrator will normally be a member of the Staff of the Biochemistry Department – often a lecturer in your course. The assistant demonstrators will be graduate students working for a PhD research degree, or post- doctoral research workers who have recent and sympathetic memories of the difficulties felt by studying the subject. Rely on them not only to sort out practical difficulties but also to help you make sure you understand what the experiments are about and what your results mean. They may well also be able to help you understand theoretical or lecture material. Discussions As the class size is relatively small, discussion of the practical work will be informal and will normally take place in the laboratory at the end of the experimental work. Demonstrators will be on hand at these occasions and you are urged to make the most of these discussions and take an active part in them. Safety and care in the laboratory PLEASE be careful in the laboratory. Attend to your own safety and that of others around you: this is a statutory obligation – a matter of law. PLEASE also extend your consideration to the equipment in the laboratory. Most of it is costly to repair or replace. Policy, rules, and guidance on safety are given on the pages in this handbook headed 'Safety in the Biochemistry Part I Teaching Laboratory'. You must read this before coming to the first practical. In addition, you must also watch the short film about lab safety before coming to the first practical, which can be found on Moodle. At the end of the safety pages, there is a declaration form to acknowledge that you have read the document and will abide by the rules set out. Copies of the form can be downloaded from Moodle, or will be available at the first practical class. This form must be signed, and will be collected during the first practical. Practical sheets You are given comprehensive notes for each practical. They are colour-coded according to purpose. The practical notes must be read before you come to the practical. In addition, there will be a ‘pre-practical’ quiz available on Moodle that can be 14 © Department of Biochemistry, 2018 BMB Introduction NST 2018-19 completed prior to the practical class, which will help you to complete your practical most efficiently. a) Green sheets The GREEN SHEETS set the context and state the learning objectives for each practical. If appropriate, they will indicate the lecture handout material that you might want to review before reading the practical notes. b) White sheets The WHITE SHEETS contain the experimental plan and instructions. These are NOT a recipe to be read for the first time when you are faced with the experiment. Make a practice of scanning the white sheets in advance, a day or two before the practical. Not to take in every detail, which will only make sense when you have the apparatus in front of you, but to get an overview of the planned experiment. Once in the laboratory, the senior demonstrator will give a brief introduction and you should then read and follow the instructions carefully. Take a few minutes to read through the instructions again at the beginning of the practical – don’t just dive in, no matter how busy everyone else looks. Always try to think out the principles of what you are doing as you go along, and to understand what is going on in the procedures you carry out. Ask the demonstrators – or they may well ask you first! Probably the most important contribution you can make to the success of your experiments is in making up reaction mixtures. THINK about what you are adding, so you add the CORRECT components. MEASURE and DISPENSE the volume of component reagents ACCURATELY using the semi-automatic GILSON PIPETTES provided. If in doubt as to how to use a Gilson properly watch the video on the course Moodle site. c) Blue sheets The BLUE SHEETS are for processing the data and also contain questions about the practical to help you understand what you have done, and why. They are a form of self- assessment – we don’t take them in and give marks, but demonstrators can advise and comment on your efforts. You will find them helpful when preparing for the end of year exam, so it is important to get them filled out properly. Going from raw numbers that come straight off an instrument to a meaningful calculated result is found quite difficult by most students, and may be tested in the examination. So take advantage of the help available. You should complete the Blue Sheets during the practical if that is feasible, or as soon after as possible after each practical. Bring your calculators to each session. Graph paper is available if you don’t have your own. Compare your results against the yellow sheets (see below) or discuss with a demonstrator. d) Yellow sheets The YELLOW SHEETS contain specimen results and, where relevant, examples of how to analyse them. They will generally be handed out either at the end of the practical or at the appropriate discussion period. 15 © Department of Biochemistry, 2018 BMB Introduction NST 2018-19 e) Pink sheets The PINK SHEETS contain important background material related to several practicals and practical aspects of biochemistry. They are put at the front of the book of practical notes as appendices to this introduction. They include an introduction to units, to pH and buffers, and to the principles of spectrophotometry. Try and have a brief look at them before your first practical. Raise any difficulties with your supervisor or a demonstrator. Recording your results You should bring your own notebook or loose-leaf book along to the practical class. In each experiment plan what observations you will make. Record your results and interpretations directly into the notebook as you go along rather than on scraps of paper that will surely get lost. Also record any arguments and conclusions from your data if there is not enough space for these on the summary sheets. You should endeavour to complete this writing up of your argument and conclusions during the experiment itself. There is no need to write out again what is already in your practical sheets. Concentrate on ensuring that you get down the key arguments and conclusions. Any writing up after the experiment is not expected to take more than about an hour for a day's practical, but it is important to do it at once while the experiment is still fresh in your mind. The results of an experiment are often best recorded graphically as well as numerically. So far as possible the graph is best drawn while you are doing the experiment as you go along, so that in doubtful regions points may be repeated or additional ones obtained. The conventional way of plotting a graph is to plot the dependent variable (the thing you measure) on the ordinate (vertical axis) against the independent variable (the thing you fix) on the abscissa (horizontal axis). Remember to label the axes with the quantity being plotted and units involved. Remember, too, that the way you plot a graph shows your interpretation of the experiment. Consult Appendix 1 on “Units” for further guidance. Your notebooks are not “marked” as part of a “summative assessment” or examination. Rather they are to help you get to grips with each practical as you do it and write it up so that you still understand it when it comes to revision later in the year. To help with this, we ask you to complete the Blue Sheets (see above). If you feel all at sea, or if your Blue Sheets are full of blank spaces make a point of asking a demonstrator for help. Moodle and the Practicals There is a significant amount of information online to support the practical classes, and it is important that you access this prior to beginning your first practical. The information you will find is as follows: A film that provides essential safety information for the classroom – this must be watched prior to your first practical class Films that provide information about practical lab techniques (E.g. use of Gilson micropipettes) Pre-practical quizzes that are available before the start of each practical class. Interactive practicals materials (new for 2018/19) – great for checking you understand the techniques used in the practical classes The various colour coded sheets described above (i.e. green, white, and pink sheets) 16 © Department of Biochemistry, 2018 BMB Introduction NST 2018-19 Technique posters that provide a ‘snapshot’ of information that describes commonly used biochemical techniques. This knowledge is typically assumed in lectures and practical classes, so use these resources to remind yourself of necessary techniques. Tutorial videos to explain biochemical methods (E.g. primer design) Materials required for the Journal Clubs (Michaelmas and Lent Terms) and the Experimental Design Exercise (Lent Term). An additional Experimental Design exercise is also included on Moodle if you want to give it a go. Journal Clubs There are two Journal Clubs, one on a molecular topic and the other more cell biological. You will be given a published paper, with some guidance notes and questions, to analyse during the week before the practical session in which you critically evaluate its merits in a small group directed by one of the Biochemistry staff. Most students find this a challenging but worthwhile exercise, since it gives exposure to the raw material of the scientific literature. Experimental Design Exercise There will be a teaching session on experimental design in Lent Term. You will be briefed in advance of the session. You will be allocated to groups and given an outline of a problem to address. You will be required to design an experimental strategy to investigate the problem and will report back as a group to a discussion session, led by a member of staff. Examinations General advice on examination skills and on the criteria used for marking and classing have been drawn up by the Faculty of Biology and the Management Committee for the Natural Sciences Tripos. They can be found by following links from these web sites http://www.biology.cam.ac.uk/exams/skills http://www.natsci.tripos.cam.ac.uk/exams https://www.biology.cam.ac.uk/exams/raven/marking-tripos Some more specific information relating to the Biochemistry & Molecular Biology examination is given below. Copies of recent past papers are provided on Moodle. Answers to the data-handling questions in Paper 3 have been made available to your supervisors; it’s better for you to try the questions before looking at the answers! Quizzes based on the data-handling questions in Paper 3 from previous exams can also be found on Moodle. General University guidance can be found at: http://www.admin.cam.ac.uk/students/studentregistry/examinations/ Reports from the Senior Examiners are also available on Moodle. 17 © Department of Biochemistry, 2018 BMB Introduction NST 2018-19 NST Part IB Biochemistry & Molecular Biology The examination consists of three papers, each of three hours’ duration, and each paper carries equal marks. Paper 1 consists of five sections, and students should answer one question from each section. Sections A and B will be based on lectures in the Michaelmas Term, Sections C and D on lectures in the Lent Term and Section E on the Easter Term lectures. Each section will carry equal marks. Paper 2 consists of two sections. Section A is made up of roughly 14 short-answer factual questions based on the whole lecture course, and carrying equal marks. Students should attempt all questions in Section A. Section B consists of four or five essay questions of a more general and wide-ranging nature than those in Paper 1, from which students need to answer two. These can be perceived as “open-ended” questions in which the student may be asked to develop an issue that was not specifically dealt with in the lectures, but is based on the material taught in the lectures. Sections A and B will carry equal marks. Paper 3 is concerned with practical techniques covered in the practical classes and journal clubs, as well as other material in the practical section of the course handbook and experimental techniques covered in the lecture course. There are three sections. Sections A and B each contain two to three questions while Section C consists of one longer subdivided question. Sections A, B, and C will carry equal marks. All questions in Paper 3 should be attempted. When answering essay questions, take particular care that you have absorbed what the question is specifically asking for. It is a common fault for candidates to react unthinkingly to a "trigger word" and simply write all they know in response. Take a little time to reflect, rather than leaping straight in. The examiners will have regard to the style and methods of candidates' answers. You should write legibly and not adopt note form unless specifically requested to do so. Helpful diagrams are welcome as part of an essay. You are perfectly free to use abbreviations that are standard scientific vocabulary without definition (E.g. G6P, ATP, DNA, RNA). 18 © Department of Biochemistry, 2018 BMB Lecture Timetable NST 2018-19 BMB Lecture Timetable 2018-19 Course Organiser: Dr Dee Scadden ([email protected]) All lectures start at 10am in the Sanger Building Lecture Theatre, Department of Biochemistry, Old Addenbrooke’s site. Entrance from Tennis Court Road (see map). Note that some lectures begin earlier in Term, and end later in Term, than is usual. This is to allow more time between the end of the course and the examinations. MICHAELMAS TERM: Genes and proteins; macromolecules in action First Lecture on Friday, October 5th; Last Lecture on Friday, November 30th Most relevant Date No. Title Lecturer Techniques Posters Introduction to the course Oct 5 Dr ADJ Scadden (in lecture 1) 1, 2, 4, 9, 13, 15, 16, 17, Oct 5, 8, 10, 5 Gene cloning and manipulation Dr ADJ Scadden 19, 21, 20, 22, 26, 28, 12, 15 29, 30, 31, 33 Oct 17, 19, Post transcriptional control of gene 5 Dr J Mata 22, 24, 26 expression Nucleic acid structure, protein- Oct 29, 31 5 nucleic acid interactions and Prof. BF Luisi Nov 2, 5, 7 transcription Nov 9, 12, Protein structure, function and 5 Dr M Hyvönen 14, 16, 19 evolution Nov 21, 23, Enzyme catalysis and protein 5 Prof. F Hollfelder 26, 28, 30 Engineering LENT TERM: Energy transduction, cell signalling and cell proliferation First Lecture on Wednesday, January 16th; Last Lecture on Friday, March 15th Most relevant Date No. Title Lecturer Techniques Posters Jan 16, 18, 21, 6 Control of metabolism Prof. KM Brindle 23, 25, 28 Jan 30, Feb 1, Energy transduction in bacteria, 6 Prof. GC Brown 4, 6, 8, 11 mitochondria and chloroplasts Feb 13, 15, Transmembrane signalling; 6 Prof. S Lummis 18, 20, 22, 25 molecules and mechanisms Feb 27, Mar 4 Control of eukaryotic cell growth Prof. DM Carrington 1, 4, 6 March 8, 11, Oncogenes, tumour suppressor Prof. GI Evan & 4 13, 15 genes and cancer Dr TD Littlewood 19 © Department of Biochemistry, 2018 BMB Lecture Timetable NST 2018-19 EASTER TERM: Biochemistry of Microorganisms First Lecture on Wednesday, April 24th; Last Lecture on Monday, May 13th, followed by practical exam discussion. Most relevant Date No. Title Lecturer Techniques Posters April 24, 26, 3 Bacterial chemotaxis Dr R Monson 29 Bacterial signalling and secretion May 1, 3 2 Prof. GPC Salmond systems May 6, 8, 10, 4 Molecular biology of protozoa Prof. DM Carrington 13 20 © Department of Biochemistry, 2018 BMB Practical Timetable NST 2018-19 BMB Practical Timetable 2018-19 Practicals are held in the Biochemistry Teaching Laboratory, Hopkins Building, Tennis Court Road, commencing at 11am, unless stated otherwise. Monday sessions commence at 12 noon. Enter the classroom from the door opposite the Genetics Department (see map at the start of this handbook). MICHAELMAS TERM 2018 Senior Most relevant Dates Practical Demonstrator techniques posters Safety Week 1 Cloning of Oct1 POU domain gene into E. 2, 15, 26 October 4 – 10 Dr D Scadden coli plasmid. Restriction mapping of plasmid. Week 2 Expression of POU domain in E. coli. Melting properties of DNA. Prof. B Luisi October 11 – 17 Week 3 Purification of POU domain Prof. N Gay 13, 1, 3, 15 October 18 – 24 Week 4 5, 7, 13, 15, 16, 18, 20, Electrophoresis mobility shift assay Dr N Standart October 25 – 31 22, 28 Week 5 Using Online Databases as Tools Dr D Scadden 13, 26 November 1 – 7 Venue: Craik Marshall Building 1.30 pm Discussion in lab of weeks 1-5: 11.00 am Dr J Rees (Monday 12 noon) Week 6 November 8 –14 Journal Club (2 pm, except Monday 3 Staff 22, 34 pm) Venue: see Moodle Week 7 Reactivity and enzyme catalysis Prof. F Hollfelder November 15 – 21 Venue: see Moodle. Week 8 Protein structure by molecular graphics Dr RW Broadhurst November 22 – 28 Venue: Craik Marshall Building, 1.30 pm 21 © Department of Biochemistry, 2018 BMB Practical Timetable NST 2018-19 LENT TERM 2019 Senior Most relevant Dates Practical Demonstrator techniques posters Week 1 Subcellular fractionation Dr M de la Roche 15 January 17 – 23 Week 2 Kinetic analysis of catalysis by chymotrypsin Dr S Weyand January 24 – 30 Experimental design: 11-1 pm (Monday Staff 3pm start) Prof. M Carrington Week 3 Venue: see Moodle January 31 – February 6 Metabolic Control in silico: 2.00-4.30pm (Monday 12 noon start) Prof. J Griffin Venue: Craik Marshall Building Week 4 Mitochondrial oxidative phosphorylation Prof G Brown February 7 – 13 Week 5 Cell signalling (1): Tyrosine phosphorylation Prof S Lummis/ Dr 15, 33 February 14 – 20 in platelets A Git Week 6 Cell signalling (1): Tyrosine phosphorylation Prof S Lummis/Dr A 15, 33 February 21 – 27 continued and discussion Git Week 7 Cell signalling (2): Thromboxane production 10, 37, 38, 1, 19, 35, Dr M Hyvönen February 28 – March 6 by platelets 28, 33 Cell signalling (2): Thromboxane production by platelets: data analysis & discussion Dr M Hyvönen Week 8 (11am; Monday 12 noon). 10, 37, 38, 1, 19, 35, March 7 – 13 28, 33 Journal Club (2pm, except Monday 3pm) Staff Venue: see Moodle EASTER TERM 2019 Senior Most relevant Dates Practical Demonstrator techniques posters Week 1 Microbial Biochemistry Prof. G Salmond April 25 – May 1 Week 2 Microbial Biochemistry Prof. G Salmond May 2 – May 8 Revision Session – see lecture timetable 22 © Department of Biochemistry, 2018 BMB Reading List NST 2018-19 Reading List For NST Part IB Biochemistry & Molecular Biology, 2018-2019 Most of these books should be available in your College library, but to give as many students as possible an opportunity to use them on a regular basis the Department of Biochemistry also keeps copies in the Part I Section of the Colman Library (in the Hopkins Biochemistry building, facing Tennis Court Road on the Downing Site). Selected books may be borrowed overnight from this collection. You are also welcome to make use of the main collection of books and journals, but borrowing these is not permitted. Part II and Part III Biochemistry students have priority for seating in the library. The Assistant Librarian is available to help locating books. Part IB BMB students who wish to use the library outside the hours of 8.30 am – 5 pm, Monday-Thursday, or 8:30 am - 4 pm, Friday, should ask the Hopkins building receptionist to programme their University proximity card for access. There are a number of very good biochemistry text books available – some of which are quite general and cover similar topics, while others are more specialized. It’s probably a good idea to primarily use the ‘General Biochemistry Texts’, then use the others to dip in and out of as required. The use of relevant textbooks is essential for underpinning the material given to you in lectures – do make the most of the resources available to you. Recommended books If you wish to buy, we suggest that you browse first in libraries, book shops or third-years’ book collections. a. General Biochemistry Texts There are good companion web sites that give further information about these books, and contain useful onward links. Biochemistry, Berg, J. M., Tymoczko, J. L. and Stryer, L. (Freeman, 8th edition, 2015). For access to Student Resources see: www.macmillanlearning.com/Catalog/product/biochemistry-eighthedition- berg/studentresources - tab Molecular Biology of the Gene, Watson, J. D. et al. (Pearson Education, 7th edition). Lehninger Principles of Biochemistry, Nelson, D. L. & Cox, M. M. (Freeman, 6th edition, 2017). Particularly useful for bioenergetics and metabolism. Good for signalling. For access to Student Resources see: www.macmillanlearning.com/Catalog/product/lehningerprinciplesofbiochemistry- sixthedition-nelson/studentresources - tab Molecular Biology of the Cell, Alberts, B. et al. (Garland Science, 6th edition 2014). The largest and most comprehensive of the cell and molecular biology textbooks, now with a CD-ROM as well as a revised Problems Book. Will be useful for Part II courses as well as IB. 23 © Department of Biochemistry, 2018 BMB Reading List NST 2018-19 www.garlandscience.com/product/isbn/9780815345244 Molecular Biology of the Cell – The Problems Book. Wilson, J. & Hunt, T. (Garland Science, 6th edition 2014). b. Books on specific topics Protein structure, function and evolution Protein Structure and Function, Petsko, G. & Dagmar, R., 2008 Enzyme catalysis and protein engineering: Introduction to Protein Structure, Branden, C. & Tooze, J. 2nd edition, 1999. Biochemistry, Abeles, R.H., Frey, P.A. & Jencks, W.P., 1992. Proteins, Creighton, T.E. 2nd edition, 1995. From Enzyme Models to Model Enzymes, Kirby, A.J. & Hollfelder, F., 2009. Structure and Mechanism in Protein Science – A guide to Enzyme Catalysis and Protein Folding, Fersht, A., 1999. Enzymatic Reaction Mechanisms, Frey, P.A. and Hegemann, A.D., 2007. Energy transduction Bioenergetics, Nicholls D.G. & Ferguson S.J. 4th edition, 2013. Molecular Mechanisms of Photosynthesis, Blankenship R.E. 2nd edition, 2014. Oncogenes, tumour suppressor genes and cancer: The Biology of Cancer, Weinberg, R. Garland Science, 2nd edition, 2014. The most comprehensive cancer biology textbook complete with a CD-ROM. Will be useful for Part II courses as well as IB. 24 © Department of Biochemistry, 2018 BMB Reading List NST 2018-19 Other sources of information a. Journals – well worth consulting Research-oriented summaries (2-4 pages) of important topics in biochemistry and molecular biology are contained in the monthly journal Trends in Biochemical Sciences (usually known as TIBS). A similar journal is Bioessays. The Scientific American usually contains at least one biochemical article per issue and is well worth a glance. Copies of these journals are found near the Part I reading area. b. Web Sites Ever more useful source of information. We shall just give here two of our favourite general addresses, which have many forward links. Online Mendelian Inheritance in Man (www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM) Swiss Institute of Bioinformatics (www.expasy.ch/) c. Links to literature sources on Moodle The Reading list and links to Textbooks and Journals can be found on Moodle in the book entitled “Course Information”. Textbooks via pubmed Links to Electronic Journals via the University Trends in Biochemical Sciences (TiBS) Current Opinions in Chemical Biology Current Opinions in Structural Biology 25 © Department of Biochemistry, 2018 Plagiarism NST 2018-19 Plagiarism As of October 1st 2018 the university has adopted the definition below: “Plagiarism is defined as submitting as one’s own work, irrespective of intent to deceive, that which derives in part or in its entirety from the work of others without due acknowledgement; or, in the case of self-plagiarism, unless explicitly permitted by regulation, submitting one’s own work that has already been submitted for assessment to satisfy the requirements of any other academic qualification, or submitted for publication without due acknowledgement. It is both poor scholarship and a breach of academic integrity.” (http://www.admin.cam.ac.uk/univ/plagiarism/students/statement.html) Although there is no assessed course work for Biochemistry and Molecular Biology it is an essential part of your scientific training that, in your supervision work and any other writings, you ensure you are following best practice regarding avoiding plagiarism. General university guidelines are available at: http://www.admin.cam.ac.uk/univ/plagiarism/students/statement.html Faculty guidelines are available at: http://www.biology.cam.ac.uk/exams/plagiarism and are reproduced below. Note that the use of essays purchased from any source or copied from other students is unacceptable regardless of whether the source is acknowledged. It is an important aspect of academic integrity to cite all sources on which you base your work (even if it is not copied directly from them), be they published in hard copy or web- based. Your supervisor may ask you to do this for written work produced during the year, and this can be useful for your subsequent revision. However, full citation is not expected in written unseen examinations such as those taken at the end of the year for BMB. If you still have questions after reading the faculty guidelines below, you should talk to your Supervisors and /or Director of Studies and/or the Course Organiser. The following guidance has been issued by the Faculty Board of Biology: In general, plagiarism can be defined as: “The unacknowledged use of the work of others as if this were your own original work” In the context of an examination, this amounts to: Passing off the work of others as your own to gain unfair advantage. Such use of unfair means will not be tolerated by the University; if detected, the penalty may be severe and may lead to disciplinary proceedings being taken against you. 26 © Department of Biochemistry, 2018 Plagiarism NST 2018-19 1. The scope of plagiarism Plagiarism is defined as submitting as one's own work, irrespective of intent to deceive, that which derives in part or in its entirety from the work of others without due acknowledgement. It is both poor scholarship and a breach of academic integrity. Examples of plagiarism include copying (using another person’s language and/or ideas as if they are a candidate’s own), by: quoting verbatim another person’s work without due acknowledgement of the source; paraphrasing another person’s work by changing some of the words, or the order of the words, without due acknowledgement of the source; using ideas taken from someone else without reference to the originator; cutting and pasting from the Internet to make a pastiche of online sources; submitting someone else’s work as part of a candidate’s own without identifying clearly who did the work. For example, buying or commissioning work via professional agencies such as ‘essay banks’ or ‘paper mills’, or not attributing research contributed by others to a joint project. Plagiarism might also arise from colluding with another person, including another candidate, other than as permitted for joint project work (i.e. where collaboration is concealed or has been forbidden). A candidate should include a general acknowledgement where he or she has received substantial help, for example with the language and style of a piece of written work. Plagiarism can occur in respect to all types of sources and media: text, illustrations, musical quotations, mathematical derivations, computer code, etc. material downloaded from websites or drawn from manuscripts or other media published and unpublished material, including lecture handouts and other students’ work Acceptable means of acknowledging the work of others (by referencing, in footnotes, or otherwise) vary according to the subject matter and mode of assessment. Faculties or Departments should issue written guidance on the relevant scholarly conventions for submitted work, and also make it clear to candidates what level of acknowledgement might be expected in written examinations. Candidates are required to familiarize themselves with this guidance, to follow it in all work submitted for assessment, and may be required to sign a declaration to that effect. If a candidate has any outstanding queries, clarification should be sought from her or his Director of Studies, Course Director or Supervisor as appropriate. Failure to conform to the expected standards of scholarship (e.g. by not referencing sources) in examinations may affect the mark given to the candidate's work. In addition, suspected cases of the use of unfair means (of which plagiarism is one form) will be investigated and may be brought to one of the University's Courts. The Courts have wide powers to discipline those found guilty of using unfair means in an examination, including depriving such persons of membership of the University, and deprivation of a degree. 27 © Department of Biochemistry, 2018 Plagiarism NST 2018-19 2. How to avoid plagiarism The stylistic conventions for different subjects vary and you should consult your Course Organiser or project supervisor about the conventions pertaining in your particular subject area. Most courses will issue written guidance on the relevant scholarly conventions and you are expected to have read and to follow this advice. However, the main points that apply to submitted work (e.g. dissertations, project reports) are: when presenting the views and work of others, include in the text an indication of the source of the material, e.g. 'as Sharpe (1993) has shown,' and give the full details of the work quoted in your bibliography if you quote text verbatim, place the sentence in inverted commas and give the appropriate reference, e.g. 'The elk is of necessity less graceful than the gazelle' (Thompson, 1942, p 46) and give the full details in your bibliography as above if you wish to set out the work of another at length so that you can produce a counter- argument, set the quoted text apart from your own text (e.g. by indenting a paragraph) and identify it by using inverted commas and adding a reference as above. NB long quotations may infringe copyright, which exists for the life of the author plus 70 years if you are copying text, keep a note of the author and the reference as you go along, with the copied text, so that you will not mistakenly think the material to be your own work when you come back to it in a few weeks' time if you reproduce an illustration or include someone else's data in a graph include the reference to the original work in the legend, e.g. (figure redrawn from Webb, 1976) or (triangles = data from Webb, 1976) if you wish to collaborate with another person on your project, you should check with the Course Organiser to see whether this might be allowed and then seek their permission if you have been authorised to work together with another candidate or other researchers, you must acknowledge their contribution fully in your introductory section. If there is likely to be any doubt as to who contributed which parts of the work, you should make this clear in the text wherever necessary, e.g. 'I am grateful to A. Smith for analysing the sodium content of these samples' be especially careful if cutting and pasting work from electronic media; do not fail to attribute the work to its source. If authorship of the electronic source is not given, ask yourself whether it is worth copying Please note that during written answers for unseen examination papers, you will not be penalised for failures to reference information in this manner. 3. The Golden Rule: The examiners must be in no doubt as to which parts of your work are your own original work and which are the rightful property of someone else. For the University-wide statement on plagiarism, and further information on the topic, please see: http://www.admin.cam.ac.uk/univ/plagiarism/ 28 © Department of Biochemistry, 2018 Synopsis of the BMB lecture course NST 2018-19 Synopsis of the BMB lecture course Note: This information is provided at the beginning of the year for your guidance and that of your supervisors. It is not intended to be a comprehensive list of contents. Lecturers will all issue their own handouts, and may vary the topics and the order in which they are presented. Michaelmas Term: Genes and proteins – macromolecules in action In Michaelmas, the course examines the molecular biology of DNA and protein structure. How is DNA packaged in cells? How does chromatin structure affect gene expression? How is genetic engineering actually carried out? How are transcription and translation regulated? What are the principles of protein design and how can we exploit them through protein engineering? § Dr D Scadden: Gene cloning and Manipulation These lectures introduce the techniques of gene cloning and manipulation that underpin much of the work described in the rest of the course. Building on material covered in the Part IA Biology of Cells lectures, we look at the use of various techniques to ask specific experimental questions. Examples of topics covered include: The polymerase chain reaction and its various applications Vectors and hosts that are used in conventional gene cloning Investigating how clones may be used experimentally. E.g. for preparing recombinant fusion proteins, making RNA for in vitro studies, etc. Methods for reducing gene expression (e.g. RNAi, CRISPR/Cas9), and for creating transgenic mice. § Dr J Mata: Post-Transcriptional Control of Gene Expression The production of functional proteins involves multiple processes in addition to transcription. Although these steps are usually referred to as post-transcriptional, many of them occur concurrently with transcription. These lectures will introduce the processes required for the formation of a mature RNA in eukaryotic cells (capping, splicing and 3’ end processing), translation (in both prokaryotes and eukaryotes) and RNA decay. The basic machinery that carries out these processes, as well as the mechanisms by which this machinery is modulated in a gene-specific manner, will be addressed. § Prof. B Luisi: Nucleic acid structure, Protein-Nucleic acid Interactions and Transcription These lectures cover the first step in gene expression – transcription of RNA using genomic DNA as template. How do RNA polymerases recognise the correct locations at which to initiate transcription, and how can this be regulated? Six main topics will be covered: DNA and RNA structure Prokaryotic transcription mechanisms Prokaryotic transcriptional regulation Packaging of eukaryotic DNA into chromatin Eukaryotic transcription – core promoter and general transcription factors (GTFs) Eukaryotic transcription – activating transcription factors and enhancers 29 © Department of Biochemistry, 2018 Synopsis of the BMB lecture course NST 2018-19 The overarching theme of DNA-protein interactions – both sequence-specific and non- specific – runs through all of these topics. At appropriate points, relevant experimental approaches and techniques will be highlighted. § Dr M Hyvönen: Protein Structure, Function and Evolution Proteins play most of the effector roles in living organisms. They maintain the structures of cells, of the extracellular matrix and tissues; they catalyze most reactions in cells and generate mechanical force in the muscles; they are involved in information transfer through recognition of other molecules and can act as ligands, as receptors, as messengers, and as transcription factors; they act as receptors, gates and channels in membranes. The aim of these lectures is to understand the unique principles of protein structure from primary structure to formation of large oligomeric complexes and molecular machines and to introduce the methods that are used to study protein structures from optical spectroscopies through X-ray crystallography and NMR to cryo electron microscopy. We will also discuss how proteins have evolved and how analysis of protein structure can help us to understand the evolutionary relationships between different proteins and their function. § Prof. F Hollfelder: Enzyme Catalysis and Protein Engineering This lecture series focuses on how the peptide and protein structures discussed in the preceding module can assume functions – and on experiments that delineate the mechanisms involved. First we develop ideas about enzyme catalysis, mechanism and kinetics. We look in detail at the co- operative (allosteric) molecular basis of metabolic regulation. Other protein structures that are discussed include immunoglobulins and their binding to specific antigens, and the principles of protein folding and stability. Finally we look at the ‘holy grail’ of protein engineering and mechanistic enzymology – how to create novel, functional proteins, by rational design, semi-rational approaches, and by directed evolution. Lent Term: Energy transduction, cell signalling and cell proliferation The course now builds on the molecular foundations laid in the Michaelmas Term to develop an integrated view of cellular processes. How do cells make a continuous supply of energy available for transcription, translation, ion pumping, biosynthesis and a host of other processes? How is metabolism regulated according to the varying needs of the cell? What are the mechanisms by which hormones regulate intracellular processes? How is normal eukaryotic cell growth controlled, and what goes wrong when such control is pathologically disturbed in cancer? § Prof. K Brindle: Control of Metabolism The aims of these lectures are: To examine the different ways in which enzyme activity may be controlled. To consider the benefits these different modes of control offer for the regulation of flux in metabolic pathways. This discussion takes place in a wider context, as these various modes of control are employed throughout biological systems. Textbook descriptions of control in the metabolic pathways tend to assume that the enzymes involved are ‘soluble’ and homogeneously distributed in the cell cytoplasm. We will see how this is not the case: rather, a high degree of spatial organisation is critical to the control of these pathways. 30 © Department of Biochemistry, 2018 Synopsis of the BMB lecture course NST 2018-19 Various experimental approaches are described for studying how metabolism is controlled, with particular emphasis on methods that may be used to study intact systems. These include: Metabolic control analysis, which allows for quantitative determination of the importance of any enzyme for flux control in vivo. Two key non-invasive spectroscopic techniques – fluorescence and NMR – that permit the study of metabolic events in intact cells and tissues. § Prof. G Brown: Energy Transduction in Bacteria, Mitochondria and Chloroplasts Bioenergetics is the study of how energy is acquired and used in living systems. Recent discoveries of key structures and mechanisms have greatly enhanced our understanding of this process. This knowledge is being applied to medicine, nanotechnology, and the energy industries, informing our attempts to develop renewable biological energy sources. The six lectures explore how bacteria, plants and animals use light, electrons, protons and ATP to transduce energy from the sub-molecular to the cellular level. The lectures use an evolutionary emphasis to make it easier to understand the diversity of bioenergetics systems in nature. § Prof. S Lummis: Transmembrane Signalling: Molecules and Mechanisms Cells are continuously bombarded by many different types of signal; the ability of these cells to respond appropriately to such signals is critical for cell survival, adaptation, and specification of function, whether they are individual amoebae or components of a large, complex organism such as a human. This lecture course explores how cells monitor the presence of specific extra-cellular signalling molecules and how these signals then instigate and drive complex and interwoven intracellular responses. The course will focus on: The diversity of signals carrying information to cells; these range from single photons and small molecules to complex proteins. The relatively few mechanisms, usually involving plasma membrane receptors, by which the cell perceives the signal. The means by which the cell decodes 'the message', a process which may be very rapid, as in neurotransmission, or much slower, as in the signals that regulate gene expression and control growth. The lectures will, for example, examine the roles of the ‘second messengers’ that often mediate part of cell signalling cascades, and will explore how these cascades allow very low concentrations of initiating signals to generate large responses in their target cells. Special attention is paid to G-protein coupled responses, and to the multiple roles played by protein phosphorylation in relaying intracellular signals. This lecture course will be complemented by two successive practical classes in which students gain hands-on experience of the techniques used to probe the roles of proteins in three different cell signalling pathways. § Prof. M Carrington: Control of Eukaryotic Cell Growth The cell cycle is the term used to describe the succession of events that occur to produce two cells from one. An understanding of the molecular events involved in progression through the cell cycle is central to solving the larger problems of how the tightly controlled expansion of cell populations during the development and growth of any organism occurs 31 © Department of Biochemistry, 2018 Synopsis of the BMB lecture course NST 2018-19 and how the loss of regulation of the cycle results in disease – not just cancer but also the inappropriate growth of normal cells. The aims of the lectures are: To give an understanding of the experimental approaches that can be taken to investigate the molecular machinery of a complex biological process. To explain how the molecular components that regulate cell cycle progression were identified and how their function was determined. To discuss a model of how the ordering of transitions that ultimately lead to cell division is regulated. § Prof. G Evan & Dr T Littlewood: Oncogenes, Tumour Suppressor Genes and Cancer The next four lectures build on the story of the cell cycle in eggs and yeasts by describing how normal mammalian cell proliferation is controlled. The focus is on the mechanisms of normal signalling pathways – growth factors and mitogens, their receptors and the mitogenic signals they generate inside the cell, and the pathways that then transduce such mitogenic signals to the various intracellular effectors that precipitate cell growth and replication. The principal effector responses to mitogenic signalling are transcriptional activation of proliferation-associated and cell survival genes and repression of growth suppressing genes, activation of RNA and protein synthesis, and an abrupt shift of metabolism to biosynthesis and aerobic glycolysis. These lectures address the question of what happens in diseases, such as cancer, where control of cell growth, proliferation, survival and migration is lost through activating mutations in proto-oncogenes, and inactivating mutations in tumour suppressor genes. This introduction to molecular oncogenesis sets the scene for a more comprehensive analysis of cancer biology in one of the Part II Biochemistry courses. Easter Term: Biochemistry of Microorganisms This final group of lectures considers bacteria and protozoa as model systems and the course now brings together the themes explored in the first two terms to examine key questions relating to prokaryotic and protist biochemistry such as motility, chemotaxis and the importance of protein targeting and other systems in virulence and pathogenicity. § Dr R Monson: Bacterial Chemotaxis and Signal Transduction The field of bacterial chemotaxis and motility encompasses perhaps the best-understood prokaryotic signalling pathway. We start by using video footage of motile E. coli cells to define the basic swimming behaviour of bacteria in the unstimulated state. We then look at how this behaviour is altered when the cells are challenged with chemostimuli, and demonstrate that the observed changes correlate with the sense of flagellar motor rotation. The altered bias in flagellar motor rotation brought about by exposure to chemostimuli causes structural changes in the architecture of the flagellar filaments, and we examine how these subtle molecular alterations can give rise to substantial changes in the behaviour of the whole cell. We also look at how the molecular components of the chemotaxis and motility apparatus of the cell were discovered, and at the techniques that have been used to piece together the complex signal transduction pathway that is involved in integrating the multiple chemosensory inputs received by the cell at any given time into a single output. This signal transduction pathway involves multiple protein components, transient protein-protein 32 © Department of Biochemistry, 2018 Synopsis of the BMB lecture course NST 2018-19 interactions, phospho-transfer events and other chemical modifications, and its workings are now beginning to be understood at the atomic level. We look at how the signalling pathway is assembled, how it works, and how its output influences the rotational bias of the flagellar motor (and therefore, ultimately, the swimming behaviour of the cell). Finally, we look at what is known about the flagellar motor itself – the world’s smallest multi-speed motor, incorporating both forward and reverse gears. The ingenious methods that have been developed to study this remarkable device are discussed, including some video footage of the motor in action. Moreover, the study of chemotaxis and motility is not simply an esoteric branch of microbiology. With the recent completion of many eukaryotic genome sequences (including the human genome), it has become clear that homologues of the chemotaxis proteins are widespread in “higher” organisms, so these findings are likely to yield valuable insights into the function of many other organisms. § Prof. G Salmond: Bacterial Secretion Systems Protein secretion mechanisms are of fundamental importance in bacteria. Prokaryotes are highly tractable model systems for analysis of the basic principles of protein targeting and the ability to manipulate such targeting can be exploited in some biotechnological processes. Furthermore, the ability to actively secrete and regulate the production of structurally diverse proteins involved in bacterial virulence is a key aspect of pathogenesis in plant and animal diseases. The lectures summarise the main protein secretion systems in Gram-negative bacteria, with examples taken from pathogens of animals and plants. Evolutionary connections between secretory machines is highlighted. The general nature of bacterial cell surfaces is discussed and the exploitation of prokaryotic surface molecules that are parasitized as “receptors” by bacterial viruses (bacteriophages) is highlighted. In the lab classes associated with these lectures, students conduct experiments on protein targeting using bacterial mutants generated via transposon insertions that can generate protein fusions. In addition, global gene regulation and intercellular chemical signaling (quorum sensing) in a bacterium that makes antibiotics are both addressed. § Prof. M Carrington: Molecular Biology of Protozoa Protozoa encompass over 60,000 species of eukaryote including many that are highly divergent from animals, there is more evolutionary diversity within the protozoa than between green plants, metazoa and fungi. The best-studied protozoans are parasites that cause diverse chronic diseases, such long-term infections provide a model for studying the complex molecular interactions between pathogen and host. Trypanosome VSGs have divergent primary, but conserved tertiary, structures to function in antigenic variation and as a protective coat on the external surface of the plasma membrane. Protozoa have evolved a number of novel strategies for overcoming host resistance to infection and, in this context, lectures will address the unusual strategies for regulation of gene expression, especially of the Variable Surface Glycoproteins (VSGs) in trypanosomes. Such studies of parasitic protozoa have provided unique insights into our understanding of basic molecular processes, for example, the structure and biosynthesis of GPI anchors in the context of cell surface architecture. 33 © Department of Biochemistry, 2018 Course Management and Student Feedback NST 2018-19 Course Management and Student Feedback The Biochemistry & Molecular Biology Course is run by a Management Committee, consisting of the course organiser and lecturers from the course. The Management Committee decides broadly on the content of the lectures and practicals, and has the responsibility for organising and delivering these. Decisions made by the Management Committee are influenced by the Consultative Committee, which comprises student representatives (chosen by you) and lecturers from the course. The Consultative Committee meets at the end of each term (dates below) and looks at the results of student feedback questionnaires as well as hearing your views about the course. The course is revised on a yearly basis in the light of comments made by you in student feedback questionnaires and by your representatives on the Consultative Committee. Dates for the Consultative Committee Meetings for BMB: Friday November 30th, 11.15am Friday March 15th, 11.15am Monday May 13th, 11.15am Minutes of the Consultative Committees and Analysis of Student Feedback Questionnaires The minutes of the Consultative Committee meetings and the results of the student feedback questionnaires can be found on Moodle. Contact Information: Contact Name Email Role Chair of the Academic issues Management Dr Dee Scadden [email protected] relating to the Committee course Organisation of meetings of the Teaching Wendy Bundy [email protected] Consultative and Administrator Management Committees Classroom Organisation of the Dr Siolian Ball [email protected] Manager practical laboratory 34 © Department of Biochemistry, 2018 BMB Safety Information NST 2018-19 Safety Information Part I Practical Classroom University of Cambridge Department of Biochemistry 35 © Department of Biochemistry, 2018 BMB Safety Information NST 2018-19 36 © Department of Biochemistry, 2018 BMB Safety Information NST 2018-19 Safety in the Biochemistry Part I Practical Teaching Laboratory These pages MUST be read before your first practical. The ‘Safety Acknowledgement and Compliance Form’ and the ‘Health Record Form’ MUST be read, then completed and signed. Please note that the forms in this handbook are for INFORMATION ONLY, and forms for completion will be provided in your first practical class. Alternatively, copies of these forms can be downloaded from Moodle in the file “Experimental Practicals – information for before you begin the course”. 1. Departmental policy 1.1 It is the overriding policy of the University and Department of Biochemistry that experimental work, whether associated with teaching or research, be done efficiently and safely. All employees, students and research workers in the Department are required to observe the provisions of the Health and Safety at Work etc. Act 1974, the Radioactive Substances Act 1993, the Ionising Radiations Regulations 1999, and the Control of Substances Hazardous to Health (COSHH) Regulations 1988, as and when appropriate. Copies of the Acts and Regulations are available in the University Safety Office; copies of the University and Departmental Safety Regulations are available in the Teaching Laboratories. Copies of the risk assessments are available at the end of the Safety Instructions. Copies of the COSHH assessments will be put up on the notice board in the practical laboratory. 1.2 Safety in the Department begins with the individual's personal responsibility. However, in addition, each member of the academic staff, each research worker, and each technician in charge of a section or a laboratory has a statutory duty to take reasonable care for persons under his/her supervision or visitors to his/her area. 1.3 It is advisable to inform the Classroom Supervisor if you suffer from any medical problem (e.g. allergies, asthma, serious hearing or sight impairment etc.) that might be aggravated by an experiment or might affect your ability to carry out an experiment. 1.4 The staff involved with practical teaching in the Part I Laboratory have ensured, so far as is reasonably practical, that students will not be exposed to risks to their health and safety. Students have a statutory obligation to protect themselves and others from hazards resulting from their acts or omissions in the laboratories. 2. General 2.1 Smoking, eating and drinking are NOT permitted in the laboratory. 2.2 Outdoor clothing, umbrellas and bags must be placed in the under-bench cupboards provided for the purpose and not allowed to obstruct gangways or bench tops. 2.3 Suitable laboratory coats must be worn (fastened up) in the laboratory and removed before leaving. Safety spectacles must be worn when carrying out all procedures, and gloves of the appropriate type must be worn when necessary. Long hair must be restrained e.g. by means of a cap, ribbon, headband or net. 37 © Department of Biochemistry, 2018 BMB Safety Information NST 2018-19 2.4 The notes for each practical should be read before coming to the practical and note taken of any safety matters. Students are NOT permitted to do any experimental work unless a supervisor (demonstrator or member of staff) is present. 2.5 The use of unfamiliar equipment (particularly centrifuges, vortex mixers, chart recorders, spectrophotometers, and oxygen electrodes) and the handling of potentially hazardous materials will be explained to students. If a student, for some reason, misses the appropriate explanation, then it is the student’s responsibility to bring the lack of knowledge to the attention of the Classroom Supervisor, so that appropriate arrangements can be made to remedy the situation. See 3.2 below. 2.6 Glassware and plastic ware that is being used, for example to make up mixtures, should be labelled with marker pen; this avoids confusion and will help the laboratory staff. 2.7 Hazard and other labels that have been fixed to solution containers must NOT be tampered with or removed. 2.8 Hand-washing facilities are available in the laboratory. Hands should be washed before leaving. 2.9 Lavatory facilities are available just outside the main entrance to the laboratory. 3. Substances and procedures hazardous to health 3.1 Where a potential hazard (e.g. certain chemicals, biological materials and procedures) exists in a particular practical, this will be discussed in the talk before the practical and details of safe working methods will be highlighted in the practical notes. 3.2 Students must NOT use unfamiliar equipment or procedures without them having been given instruction. All safety instructions given in the preliminary talk and practical notes must be adhered to. 3.3 A hazard assessment (COSHH assessment) has been prepared for each practical; these are available for reference on the notice board situated to the right of the doorway to the NST IB classroom and exit. Extra copies of the COSHH assessments for all practicals will be made available either prior or during each practical session. 4. Waste 4.1 Broken glassware must be placed in one of the special bins provided in the laboratories where the sign 'BROKEN GLASS DISPOSAL POINT' is displayed. It must NOT be put into any other waste container. 4.2 Ordinary waste (e.g. tissues, sealing film and its backing paper, gloves) should be placed in one of the waste buckets provided at the ends of the benches. It must NOT be put into the bins for broken glass (referred to in 4.1 above). 4.3 Used plastic pipette tips must be placed in the appropriate labelled container on the bench. 4.4 Certain waste materials (e.g. used lancets, syringe needles, particular chemicals) that need specific disposal methods must be placed in the appropriate containers 38 © Department of Biochemistry, 2018 BMB Safety Information NST 2018-19 provided during that practical. They must NOT be placed in any other waste container. 4.5 No liquids should be poured down the sinks. Liquids should be disposed of in the disposal vessels supplied. 4.6 Benches should be left as waste-free and tidy as possible at the end of each practical – this reduces potential for accidents and spillages and is of considerable help to the laboratory staff. 5. Accidents 5.1 All accidents and spillages, including any personal injuries and damage caused to equipment, must be reported as soon as practicable to the Senior Demonstrator and Laboratory Manager or any of the other technicians. They will organise first aid if required and attend to the required written report. 5.2 Spillages must be cleared up immediately and the area decontaminated; they must NOT be left as a hazard to others. 5.3 A first aid box is located in the Classroom Supervisor’s office area; see the classroom plan in the practical manual. Consult a Demonstrator or member of the Technical Staff if you need items from the First Aid Box. 6. Evacuation procedures 6.1 Any fire must be reported immediately to a technician or demonstrator. 6.2 A two-tone siren means evacuate the building immediately, by the designated routes (see plan). 6.3 In the event of fire do not panic, hesitate, or run; follow any oral instructions immediately and leave in an orderly manner. Do NOT use the lift. 6.4 The designated fire exits at both ends of the laboratory have clear signs and are shown on the plan of the laboratory. Students must identify those exits on their first visit to the laboratory. 7. Summary Safe working is an attitude of mind, and the result of the sensible application of acquired knowledge. Always plan experimental work with safety in mind. Anticipate possible hazards and seek to minimise them, but be alert for the unexpected. Students should learn to carry out their work safely and maintain their working area in a safe condition, for the benefit of themselves and others working in the area. If in Doubt – ASK!! 39 © Department of Biochemistry, 2018 BMB Safety Information NST 2018-19 Please note that the ‘Safety Acknowledgement and Compliance form’ and the ‘Health Record form’ in this handbook are for INFORMATION ONLY – forms for completion will be provided in the first practical class. Safety Acknowledgement and Compliance Form NAME ________________________ COLLEGE ______________ (please print) BENCH/GROUP No ________________ (This is the number on your side of the card at the centre of the bench). PRACTICAL DAY ____________________ I have read the document entitled Safety in the Biochemistry Part I Practical Teaching Laboratory. I agree to abide by the rules contained therein. Signed ____________________________ Date ________________ This form to be handed to Laboratory Manager or her deputy Form received by Date 40 © Department of Biochemistry, 2018 Health Record Form – Record of Hazardous Substance Usage The COSHH Regulations require all individuals wo

BMB Course Handbook 2018-19 PDF

Document Details

Tags

Related

Summary

Full Transcript

Upgrade to continue