The aims of this module are:
• To provide practical skills training in a range of modern biological techniques principally based around molecular biology.
• To provide training in maintaining laboratory records and in writing scientific research papers.
On completion of this module, students should be able to:
• Demonstrate a high-level understanding of and practical competence in a range of experimental techniques that underpin modern molecular life sciences including: plasmid DNA preparation and quantification; subcloning; PCR; site directed mutagenesis; DNA sequencing; recombinant protein expression; SDS PAGE and western blotting; protein purification and analysis.
• Maintain a detailed and accurate laboratory notebook recording the experimental procedures and results obtained during the practical sessions.
• Present and critically analyse the results obtained in the form of a short scientific paper.
This module is an extended practical investigation in the form of a mini-project. The aim is to sub-clone the gfp (green fluorescent protein) coding sequence into an expression vector, express the protein, purify it and then analyse some of its properties. To achieve this, in pairs, you will work through the following experimental stages:
1. Sub-clone the coding sequence of gfp DNA from pET23 into the pET28c protein expression vector (practicals 1-3). Plasmid maps for pET23 and pET28c are included in Figure 1.
2. Transform the plasmid into E. coli DH5a cells and select by plating on LB medium containing kanamycin (antibiotic) followed by direct colony PCR to detect the presence of the gfp insert (practicals 3-4).
3. Generate 1 spectral variant (mutation) of gfp using site-directed mutagenesis and characterize by DNA-sequencing. Overall, this will yield 2 pET28c recombinants, one carrying the wild-type gfp construct and the other carrying (different) mutated gfp constructs (practicals 5-6).
4. Isolate plasmid DNA derived from each of these recombinants and transform into the expression host BL21(DE3) pLysS (practicals 6-7).
5. Induce GFP protein expression (including mutants) using the auto-induction method and assess protein expression by SDS-polyacrylamide gel electrophoresis followed by western blotting, using HisprobeTM-HRP (practicals 8-9).
6. Purify the GFP proteins using Ni-NTA chromatography (practical 10).
7. Analyse the purified proteins by mass spectrometry and fluorometric analysis (this will be done for you and the data returned to you to analyse: practical 11).
An overview of this mini-project is summarized in Table 1.
TEACHING AND LEARNING METHODS
This module will be delivered by laboratory training and experimental practice over a weekly (12 weeks) daylong sessions. You will be required to keep an individual contemporaneous record of procedures and results in the form of a laboratory notebook, which should be available for inspection by the module teaching staff during every practical session. The results of the investigation will be written-up in the form of a short research paper (in the style of FEBS Letters) at the end of the practical.
You are expected to come to each practical having read the protocol of the week and the preparatory (background) reading associated with each practical. This preparatory reading will include some questions intended to assist you in your understanding. These should be completed prior to attending the classes. The demonstrators will go through the answers during your practical session. End of module test questions will be derived from these.
This module will utilize and develop skills introduced in the Advanced Biomolecular Technologies module. Therefore, the lectures and seminars delivered as part of Advanced Biomolecular Technologies form part of the essential background material to this practical module and have been deliberately timetabled to precede the practical skills you will be using. For an overview, see Table 1.
This module will be led by Dr Chi Trinh along with the laboratory technical staff and demonstrators.
If you have any queries relating to this module, please contact Dr Trinh.
The companion textbook to the techniques covered in this module (and in BIOL5272M Advanced Biomolecular Technologies module) is:
Divan, A & Royds, J. (2013) Tools and Techniques in Biomolecular Science. Oxford University Press. Oxford.
If you are unfamiliar with the basic principles of gene cloning, then the textbook below will be useful:
Brown, T.A. (2010). Gene Cloning and DNA Analysis. 6th Edn. Blackwell Publishing.
For an overview of recombinant protein production, refer to the following:
Zerbs, S., Frank, A.M., Colart, F.R. (2009). Bacterial systems for production of heterologous proteins. Methods in Enzymology, 463, 149-168.
Divan, A & Royds, J. (2013) Tools and Techniques in Biomolecular Science. Oxford University Press. Oxford. CHAPTER 7 (sections 7.1-7.3).
For background to GFP structure, properties and uses:
Tisein, R. (1998). The green fluorescent protein. Annual Reviews of Biochemistry 67, 509-5044.
Zimmer, M. (2002). Green Fluorescent Protein (GFP): Applications, Structure, and Related Photophysical Behavior. Chem. Rev., 102, 759–782
Chalfie, M. (2009). "GFP: Lighting up life". Proc Natl Acad Sci U S A. 106, 10073–10080.
Figure 1b: Novagen pET-28a-c(+) expression vector. The vector carries an N-terminal
Work should be handed in to the Graduate School Office, marked for the attention of Dr Chi Trinh. An identical copy of the work should also be submitted electronically to the assignment upload area in the BIOL5373M module area on the VLE by the same date.
All work should be submitted to the Student Education Office marked for the attention of the module manager and an identical copy uploaded into an assessment upload area of the module in the Blackboard VLE by the same deadline. A signed Declaration of Academic Integrity Form should be attached to all assessed work at the time of submission.
INFORMATION ON PLAGIARISM
The University gives clear guidelines on what is deemed plagiarism or fraudulent work and treats this very seriously. The University has its own website: http://www.lts.leeds.ac.uk/plagiarism/ which gives advice and guidance to students – please use it and also read the Masters Bioscience Programme Handbook which also gives details on plagiarism and cheating.
LABORATORY SAFETY INFORMATION
• All students working in the Garstang Laboratories must familiarise themselves with the laboratory rules set out below.
• COSHH assessments for individual experiments are on the VLE and should be read prior to attending the practical.
• You will be provided with a laboratory coat to wear during practical work. Outdoor clothing, bags, cases etc cannot be taken in to the laboratory. These must be left in the lockers provided. You will need a 30mm padlock to secure your belongings. This can be purchased from any supermarket or a hardware store.
General Laboratory Safety Rules
1. Always wear a laboratory coat, properly fastened.
2. Do not smoke, eat, drink or apply cosmetics in the laboratory
3. Suitable eye protection and gloves must be worn in the laboratory when instructed
4. No mouth pipetting is to be carried out
5. Be conscious of hazards. Read the safety notes for each experiment. Report any accidents however slight to your Demonstrator.
6. Dispose of all broken glass, pasteur pipettes etc into “sharps” containers provided. Never put them into soft paper waste bins built in under the benches.
7. Most solutions can be safely disposed of down the sinks. Run plenty of water down after them. But some waste solvents must NOT be put into the sink; pour these into the special waste containers provided.
8. Keep your area of bench tidy and organized whilst you are working. Mop up spills at once, first consulting the demonstrator if any hazard is involved.
9. Make sure that you leave your area of the bench CLEAN and TIDY at the end of the practical.
Mobile phones/ipods/computers/ipads and similar devices should be left in your locker for the whole practical session.
Once you have quantified your plasmid DNA, you will “cut out” the gfpuv gene from the pET23 vector by setting up a restriction digest using the two restriction enzymes NdeI and HindIII. To do this, you will mix 15µl of your plasmid DNA with the restriction enzymes, NdeI and HindIII and restriction enzyme buffer. The buffer provides the optimal Mg2+, NaCl and pH conditions for the enzymes to work effectively. Restriction enzymes are extremely temperature-sensitive and therefore you should ensure that all your components (enzymes, buffer and plasmid DNA) are kept on ice until you are ready to incubate the reaction mix. The reaction mix will comprise a total reaction volume of 50µl and will be incubated at a temperature of 37°C for a minimum of 4 hours.
Experiment 1.3 Measuring plasmid DNA concentration and purity using UV spectrophotometer
You will determine the quantity and quality of the DNA by reading the optical density (OD) of your sample at 260nm and 280nm (also referred to as A260 and A280).
The reading at 260nm is used to calculate the concentration of nucleic acid in the sample. For pure, double stranded DNA: 1 OD at A260 = 50µg/ml (for a 1.0cm path length cuvette).
The ratio between the readings at 260nm and 280nm (A260/A280) provides an estimate of the purity of the nucleic acid. Pure preparations of DNA have an A260/A280 value between 1.7-2.0.
1. Read the protocol carefully and draw a flow diagram to show the main stages of the experiments.
2. Read through the COSHH form associated with this practical which is available on the VLE.
For a general overview on gene cloning consult:
Divan & Royds (2013) Chapter 1 or Brown, T.A. (2010) - see Reading List
NOTE: there are no pre-practical questions associated with practical 1.
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