BCMB30010 sem1 2021 Manual Part1 Introduction PDF

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Summary

An introduction to the practicals undertaken in BCMB30010, created after the revitalisation of the course. This contains all new practicals...

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BCMB30010 ADVANCED TECHNIQUES IN MOLECULAR SCIENCE

Practical Manual Semester 1, 2021 THIS COMPILATION OF STUDENT READING MATERIAL HAS BEEN MADE IN ACCORDANCE WITH THE PROVISIONS OF PART VB OF THE COPYRIGHT ACT FOR THE TEACHING PURPOSES OF THE UNIVERSITY. FOR USE ONLY BY STUDENTS OF THE UNIVERSITY OF MELBOURNE ENROLLED IN THE SUBJECT: BCMB30010

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BIOCHEMISTR AND MOLECULAR BIOLOGY

Name: Prac. Day: Group:

INDEX Page Message from the Head of Department .................................................................................. 3

SECTION I: GENERAL INFORMATION Staff List …………………………………………………………………………………………………. 4 Practical Timetable……………………………………………………………………………………… 5 Submission Dates………………………………………………………………………………………. 6 Subject Objectives and Organisation ………………………………………………………………… 7 Assessment.................................................................................................................................. 9 Laboratory Safety........................................................................................................................14 Laboratory Instruments,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,. 15 Cleaning and Waste Disposal .................................................................................................... 16

SECTION II: EXPERIMENTAL Experiment 1:

Mutagenesis studies of a Protein Tyrosine Phosphatase (PTP).................. 17

Experiment 2:

Recombinant expression of unmutated and mutated PTPs......................... 50

Experiment 3:

Subcellular Localisation of PTP variants (mammalian cell culture)………..151

Experiment 4:

Proteomic analysis of serum proteins using mass spectrometry…………..185 MS Bioinformatics Tutorial…………………………………………………….. 209

ACKNOWLEDGMENTS: This manual was prepared and assembled by Izabela Szambelanczyk Orval, Department of Biochemistry and Pharmacology, University of Melbourne. Text was written by Izabela Szambelanczyk Orval, Dr Leon Helfenbaum, Mrs Beverley Bencina, Assoc. Prof Heung-Chin Cheng and Prof Trevor Lithgow.

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March 2021 Dear Students, The molecular life sciences are central to biomedical sciences and biotechnology and this subject will introduce you to techniques used in molecular science and allow you to learn the theory behind these methods. These technologies are commonly used in research and in the biomedical and biotechnological industries and the subject will provide valuable training for both your future employment as a biochemist and your studies within B.Sc. Honours and graduate research programs in biochemistry and other related disciplines. The experiments carried out in this subject will include, recombinant protein expression and purification, mammalian cell culture and protein targeting and proteomic analysis of proteins using mass spectrometry. These are all techniques in use in our research laboratories and we encourage anyone who is interested in learning more about the science undertaken within the laboratories of the Department of Biochemistry and Molecular Biology at the Bio21 Institute to contact us, so that we can arrange visits and meetings with key laboratory personnel. You should also take the opportunity to use any quiet time during experiments to discuss the research activities of the Department with demonstrators supervising the practical classes. This will enable you to appreciate the wider applications of techniques being taught and will provide a chance to become familiar with research areas that may be of interest to you. Please feel free to see me if you encounter any difficulties or require assistance with the planning of your careers. I hope that you find the subject rewarding and I wish you well in your studies. Yours sincerely,

Ian van Driel Professor and Head Department of Biochemistry and Pharmacology Department of Biochemistry and Pharmacology The University of Melbourne Victoria 3010 Australia T: +61 3 8344 -2321 E: [email protected] W: http://www.biochemistry.unimelb.edu.au/2

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General Information

GENERAL INFORMATION Teaching and Laboratory Staff Coordinators Izabela Szambelanczyk Orval Amber Willems-Jones

Preparation Room Staff Brent Smith Lynn My Tran Jordan Lilley

Demonstrators: Alex Rey Brett Drummond Kim Ia Renu Shankar

Location of classes The Biochemistry and Molecular Biology Department is located in Bio21 and in the Tri-Radiate Medical Building with the Teaching Unit located on Level 3 West of the Medical Building Practical classes will be run on Level 2 of the Western Edge Biosciences (WEBS) Building. The building is associated with the faculties of Medicine Dentistry and Health Sciences (FMDHS), the Faculty of Veterinary and Agricultural Sciences (FVAS) and the Faculty of Science

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General Information

PRACTICAL TIMETABLE SEMESTER 1, 2021 Classes will commence in the first week of semester. Weeks highlighted in blue will be online classes conducted via Zoom. Remaining weeks will be faceto-face classes in the WEBS building WK

DATES

EXPERIMENT

PRACTICAL CLASSES

1

March 1, 2, 3 and 4

Experiment 1: Day 1

Mutagenic PCR

2

March 8, 9, 10 and 11

Experiment 1: Day 2

DpnI treatment & transfection

3

March 15, 16, 17 and 18

Experiment 1: Day 3

Plasmid miniprep, RE digestion. Agarose Gel electrophoresis

4

March 22, 23, 24 and 25

Experiment 2: Day 1

5

March 29, 30, 31 and 1

Experiment 2: Day 2

Induction of proteins Affinity purification, Phosphatase assays SDS-PAGE of purification Bradford protein assay.

MID-SEMESTER BREAK Western transfer, Immunoblot probe, Transfection frequencies

6

April 12, 13, 14 and 15

Experiment 2: Day 3

7

April 19, 20, 21 and 22

Experiment 2: Day 4

Enzyme activity calculations Class Discussions

8

April 26, 27, 28 and 29

Experiment 3: Day 1

Cell culture; Transfection and Fixation

9

May 3, 4, 5 and 6

Experiment 3: Day 2

10

May 10, 11, 12 and 13

Experiment 4: Day 1

11

May 17, 18, 19 and 20

Experiment 4: Day 2

MS; Collect and desalt peptides

12

May 24, 25, 26 and 27

Experiment 4: Day 3

MS; Data analysis, Class Discussion

Cell culture; imaging. Analysis of plasmids for TC48/45 variants MS; Alkylation, Reduction, Trypsin Digest

~ Highlighted Weeks will be an Online Session via Zoom ~

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General Information

SUBMISSION DATES SEMESTER 1, 2021 Laboratory notebooks will be submitted 2 days after your prac class each week. Bioinformatic Reports will be due on your prac day at the end of the practical class

WK

DATES

LAB NOTEBOOK

1

March 1, 2, 3 and 4

Week 1 – 2 days after prac class

2

March 8, 9, 10 and 11

Week 2 – 2 days after prac class

3

March 15, 16, 17 and 18

Week 3 – 2 days after prac class

4

March 22, 23, 24 and 25

Week 4 – 2 days after prac class

5

March 29, 30, 31 and 1

Week 5 – 2 days after prac class

BIOINFORMATIC REPORTS

Bioinformatics Report 1

MID-SEMESTER BREAK 6

April 12, 13, 14 and 15

Week 6 – 2 days after prac class

7

April 19, 20, 21 and 22

Week 7 – 2 days after prac class

8

April 26, 27, 28 and 29

Week 8 – 2 days after prac class

9

May 3, 4, 5 and 6

Week 9 – 2 days after prac class

10

May 10, 11, 12 and 13

Week 10 – 2 days after prac class

11

May 17, 18, 19 and 20

Week 11 – 2 days after prac class

12

May 24, 25, 26 and 27

Week 12 – 2 days after prac class

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Bioinformatics Report 2

General Information

SUBJECT OBJECTIVES AND ORGANISATION Aims The aims of this subject cover the following essential components of biochemical laboratory practice: • To provide safe practical experience in a variety of techniques • To give instruction in the correct methods for keeping scientific records and scientific report writing • To provide experience in simple experimental design and critical analysis of data • To further students' knowledge of bioinformatics searches • To assist students in evaluation of the scientific literature and to develop skills in the presentation of scientific literature. We assume a knowledge and understanding of the theory and practical techniques including calculations of dilutions and stoichiometry taught in second year Biochemistry or equivalent courses. Please retain your Laboratory Manuals from BCMB20005 ‘Techniques in Molecular Science’ as a reference. International Students and students who have transferred from other universities will be able to consult parts of the Laboratory Manuals for BCMB2005 ‘Techniques in Molecular Science’ on the LMS to see what has been covered. Some copies may be available.

Objectives On completion of BCMB30010 ‘Advanced Techniques in Molecular Science’ students will be expected to have: • Gained hands-on experience in a variety of techniques, generated results for analysis and demonstrated an understanding of the principles behind the various techniques and any safety issues encountered with these techniques • Understood the design of experiments and the roles of controls • Analysed experimental data using spreadsheets and bioinformatic resources • Kept a complete and accurate record of techniques and results obtained and used this record in the preparation of a scientific paper

Subject Outline and Organisation This subject is designed to introduce you to some of the experimental techniques commonly used in the in vitro manipulation of DNA and to the use of recombinant DNA expression systems, a basic tool in genetic engineering. The growing importance of Mass Spectrometry as an analytical tool will be demonstrated in an experiment involving the isolation and identification of a serum protein. The practical component is subdivided into four experiments each lasting several days, and each assessed separately. Students will work individually within pairs.

The first experiment requires you to mutagenize some conserved key elements of the catalytic site of protein tyrosine phosphatases.

The second experiment requires you to express the mutated (and unmutated) protein tyrosine phosphatases in a recombinant protein expression system and analyse them with enzyme assays, SDSPAGE and Western blotting. Where appropriate, data will be analysed using Excel spreadsheets. The third experiment will introduce you to aspects of mammalian cell culture. We will passage cells and prepare them for transient transfection using fusion proteins between EGFP and variants of protein tyrosine phosphatases that have been targeted to different cellular compartments. We will investigate how fluorescence microscopy can indicate the sub-cellular localisation of proteins.

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General Information

The fourth experiment will use mammalian serum as an example of a tissue containing a mixed population of proteins. By employing acrylamide electrophoresis to separate the proteins and trypsin digestion of an isolated band, mass spectrometry (primarily, MS/MS analysis) will be used to identify the protein selected and the mammalian species from which the serum was derived. In addition, students will work together in small groups with the help of lecturing staff of other third-year Biochemistry subjects as well as laboratory heads from Bio21, to analyse a scientific paper. They will present their findings to the class.

Laboratory Notebooks A laboratory notebook will be kept as a daily record of experimental work in each of these experiments and should always be available for inspection. During the first practical class, you will learn how to use Lab Archives and how to put your laboratory notebook together. Documentation in a Lab Notebook is extremely important. Keeping a accurate record of work performed is essential. Remember: “If it isn’t written down, it wasn’t done”

Biochemical Calculations Students are expected to be able to perform calculations involving dilutions and concentrations. There will be one compulsory section on biochemical calculations in the final examination. If you have problems with calculations, you can get help by consulting: • Biochemical Calculations by Irwin H. Segel and • Fundamentals of Biochemical Calculations by Krish Moorthy Both are available for overnight loan from the Laboratory Preparation Room or the Brownless Biomedical Library.

Text books and Reference material No single text completely covers this subject. A list of useful references is included below. 1.

Wilson and Walker’s Principles and Techniques of Biochemistry and Molecular Biology (2018), [8th Edition, edited by Hofmann, A. and Clokie, S.] Cambridge University Press is recommended to all students intending to continue to Honours or Masters in Biochemistry and Molecular Biology or to others planning a career in this field. It covers a wide range of topics in practical Biochemistry and Molecular Biology.

2.

Many of the experimental procedures used in this subject have been adapted from Molecular Cloning: A Laboratory Manual (1989) [2nd Ed, edited by Sambrook, J., Fritsch, E. F., and Maniatis, T. Cold Spring Harbor Laboratory]. This text (3 volumes) is available for consultation only in the Teaching Laboratory.

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Chapter 9 in a previous edition of Molecular Cell Biology (2003) [5th Ed., edited by Lodish H. et al., Freeman], or the corresponding chapter in other editions, provides background information on DNA Technology.

Readings on Line on the BCMB30010 homepage on the LMS For all Experiments and for some techniques used in these experiments, specific scientific journal articles are cited in the text. A collection of the most important of these will be available to students through ‘Readings on Line’ which can be accessed through the BCMB30010 LMS homepage.

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General Information- Assessment

ASSESSMENT There are 5 components of assessment in this subject: i.

Continuous assessment of laboratory skills and Lab notebook maintenance

50%

a. Practical laboratory skills – 20% b. Laboratory Notebook maintenance – 30% ii.

Bioinformatics/ Database Searching: Successful completion of both is a hurdle requirement

iii.

Research Report components: Written throughout semester

15%

iv.

Research Report: Final Version

20%

v.

A 1-hour exam to be held in the examination period.

15%

Total Assessment

100%

Further notes on assessment As the bulk of assessment for the semester is continuous, the following guidelines are presented to help all students appreciate what is required.

i.

Practical laboratory skills (20%)

A student must attend all classes, arriving punctually and prepared to carry out the procedures assigned for that day. Students will also be required to successfully complete the tasks assigned within the time of the practical class and maintain a complete record of the experimental details and results in the laboratory notebook. Students are expected to work in an orderly and safe manner. Efficiency in the organisation of time and the technical skills of the student will be considered. It is expected that students will demonstrate that they understand the reasons behind the operations carried out and show some understanding of the theoretical concepts underlying the experiments. To help with this understanding, it is expected that students will consult the demonstrators or the scientific literature to clarify any problems. Students are also expected to perform simple calculations involving dilutions and concentrations An Honours standard requires that additional skills be demonstrated. It is expected that students should demonstrate a detailed understanding of the reasons behind the operations that they carry out and show a thorough understanding of the theoretical concepts underlying the experiments. A high level of effectiveness in the organisation of time and a high level of technical skills will be necessary to attain an Honours standard.

ii.

Laboratory Notebook Maintenance (30%)

The following notes indicate the importance of laboratory notebooks and the way they should be kept. 1 The primary purpose of the notebook is to serve as an accurate record of experimental work from which data can be retrieved and used for report writing or repeat experiments, weeks or even months later. To this end it is essential to head each entry with the date and name of the experiment being carried out indicating clearly the methods used (e.g. page number(s) in Laboratory Manual). Label figures and tables of results clearly; a list of Absorbance readings with no indications as to the wavelength used or the nature of the samples measured is useless for subsequent interpretation. 2.

Any material NOT covered by the Practical Manual should be included in the lab notebook. This could include actual modifications to listed procedures.

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General Information- Assessment

Your lab notebook should include: • summary of techniques used each day and any variation in method from that given in the Laboratory manual • calculations for dilutions or determination of concentrations • observations and data such as plate-reader readings, photographs and descriptions of electrophoresis gels etc • comments on the quality of the data where appropriate e.g. consistency or replicates, the quality of the calibration curves, (what the r2 readings indicate), the consistency of the lanes in a gel • calculations of results • comments on results and conclusions drawn •

The record kept must be up-to-date, accurate and comprehensive.

•

results should be entered immediately they are obtained.

•

As students often work in pairs, BOTH members of the pair must record each day’s results before leaving the laboratory.

After each section of experimental work, you should enter brief comments in your notebook on your results and their significance. Some guidelines and questions to assist you in evaluating the results are presented in the relevant sections of the manual.

iii.

Bioinformatic Tutorial Reports (hurdle requirement)

For both the first and the fourth practical you need to complete worksheets dealing with the bioinformatic material for that practical. These will provide you with an understanding of the various tools used to analyse gene and protein sequences. You should complete these on the Report Sheets provided. For the first report, there is time during the allocated class in week 1. For the second report there is some time in the practical class during weeks 9 and 10. At these times there is assistance from the demonstrators. Students ...