Protein Engineering Laboratory Project

GUIDELINES FOR WRITING LETTERS STYLE REPORT

A major assessed component of this module is to write up your research findings in the form of a short scientific research paper in the style found in the journal FEBS Letters. This journal is available electronically through the University Library and you are strongly advised to familiarize yourself with the format of Letter publications before writing your report. Please also consult the lecture material you have been provided on writing research papers.

Your report must be word-processed and the length should not exceed 3,000 words (excluding figure legends, references and tables).

The structure of the report

Your report should include the following:

Title - This should be short and descriptive stating in a clear and concise manner the basis of the report. Try to avoid starting titles with general words such as The.....or A...

Abstract - This should be a maximum of a 100 words that briefly outlines the purpose of the work, clearly states the aims, and outlines the main methods, main results and the conclusions. It is usual to write the abstract LAST, once the remainder of the report or paper is completed so that it truly reflects the content. It is the first (and often only) part of the paper a potential reader will look at. It needs to convey the key message of your paper and entice the reader to read further.

Key words - A maximum of six key words not in the title should be selected. These are used by journals for indexing purposes.

Introduction - This should include a review of published literature so that the reader is aware of the current state of knowledge on the topic and can understand the theory of and/or background to the investigation. It should state clearly what problem you are addressing i and why (the purpose/aims of the experiments) and may contain a hypothesis. This section is typically 1/5th of the report.

Materials and Methods - This is a much-shortened version of the protocols detailed in the lab manual and should include the sources, reagents, cells etc. used during your work. The methods should be written as paragraphs of text and sub-headed for clarity. This section is typically a ¼ of the report. Materials and methods describe concisely but in sufficient detail for other scientists to reproduce the results you have presented in your paper.

Results - This is a summary of your main findings. Do not discuss your results at this stage, describe them only. Again use sub-headings and illustrate with appropriate figures and/or tables.

Figures and Tables
You are required to present as part of the results a maximum of 5 figures and/or tables. Each Figure and/or table must have a number, title and a descriptive legend (maximum of 50 words). The legend should be concise, yet sufficiently detailed so that figures/tables can be understood without detailed reference to the main text of your article. Figures should be numbered sequentially as they appear in the text. Similarly Tables should be numbered sequentially. You may want to consult FEBS Letters for examples of table/figure layouts.

Discussion - The discussion is a critical evaluation of your results and comparison with other published experimental and theoretical work. It may include limitations of the current work and recommendations for future study.
This is an explanation/interpretation of your results and should include the following:

• The main findings restated - this information is necessary to interpret the results
• A comparison of your results with the results of other published studies
• How your results relate to theory and accepted principles dealing with the topic
• Limitations of the study
• Implications of the study (i.e. possible applications of the results)
• Recommendations for further work

The discussion should conclude with a paragraph that summarises your main conclusions.

References - The list of references is included at the end of the paper. Reference to journal articles should contain the surname and initials of all authors, year of publication in parenthesis, full title of the paper, abbreviation of the journal title according to current practice, volume number and inclusive page articles. References to books should include the surnames and initials of author(s), year of publication, title of the book, name and initials of editor(s) in parenthesis, volume, edition, inclusive page numbers and name and location of the publisher. You may use EndNote to compile your reference list.

Workbook Outline

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, you will work in pairs 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).

Study Figure 1a for a restriction map of plasmid pET23-gfpuv showing the location of restriction sites. In this experiment, you will digest 25µl of pET23-gfpuv DNA with 40 units of the restriction enzymes Nde1and 30 units of the restriction enzyme HindIII in a total reaction volume of 50µl.
1. The restriction digest has been prepared for you in a sterile microfuge tube containing the following reagent shown in this table. (Note the restriction enzymes are added last to the PCR tube).
20 µl sterile distilled water
5 µl 10x restriction enzyme buffer
20 µl plasmid DNA
2 µl NdeI enzyme (20U/µl)
3 µl HindIII enzyme (10U/µl)

50 µl final volume
2. For the digestion leave the PCR tube in the PCR machine at 37oC for 4 hours. This will be done for you.

PRE-PRACTICAL QUESTIONS: PRACTICAL 1
1. What does sub-cloning mean? Explain this in the context of the experiments you will be carrying out during practical 1-4.

2. Study the plasmid vectors maps of the pET23 and pET28c vectors (Figure 1a and 1b of this workbook):

Why is the internal Nde1 site in the gfpuvcoding sequence deleted in the pET23gfpuv plasmid?

Why is a new Nde1 site introduced?

Why not use EcoR1 as a restriction enzyme for the restriction digest (instead of Nde1)?

3. The gfpuv gene will be restricted (cut) from the pET23 plasmid using the two restriction enzymes NdeI and HindIII.

What are the recognition sequences recognised by these two enzymes?

What types of ends are produced by these two restriction enzymes when they cut DNA?

4. How many fragments will be produced by digesting the vector pET23-gfp with the restriction enzymes NdeI and HindIII? What will their sizes be? Assume for the purpose of this question that your digestion will be complete (i.e. that all of the DNA will be fully cut by these two enzymes).

5. What would be suitable controls to use when setting up a restriction digest to show that each of your restriction enzymes is active and able to cut the DNA? (1 mark)

Experiment 2.1: Preparing agarose gels
1. 1 gram of agarose has been addeddirectly to a bottle containing 100ml of Tris-acetate buffer (TAE). The final agarose concentration will be 1% (w/v). The bottle is then placed into the microwave with the lid loosen and heated to dissolve the agarose.
2. When the agar has completely dissolved, remove from the microwave and set it aside to cool to 60°C. Check the temperature with a thermometer.
3. Meanwhile: prepare your agarose gel apparatus by positioning the plastic moulds at each end. Then position the comb in its designated slot. A demonstrator will show you how to do this.
4. When the agarose has cooled to 60°C, add 10μl of 5mg/ml Gel Red (a derivative of ethidium bromide) solution.
5. Wearing gloves (Gel Red is a safer derivative of ethidium bromide, but still demonstrates mutagenicity at the concentrations found in the stock solution).
6. Swirl the solution gently to mix, then pour 50 ml carefully into the gel mould without splashing. Two groups should use this 100ml to make one gel each.
7. Allow the gel to set for at least 30min.

Experiment 2.2: Analysing your restriction digest products by agarose gel*Refer to the laminated card provided that gives instructions for the loading and running of agarose gels*
1. Carefully remove the comb from the 1% agarose gel by easing it out from one end.
2. Carefully remove the moulds from either ends of your gel and then transfer the gel (still on its glass plate) to the electrophoresis tank. Remember to place it in the correct orientation with respect to the electrodes so that the DNA samples will migrate from the wells into the main body of the gel when the power is switched on. (Think: what charge does DNA have?)
3. Now carefully pour all your TAE buffer so that the surface of the gel is covered by about 2mm.
4. Add 10µl of 6x DNA loading buffer to your restriction digested plasmid and also to the uncut plasmid sample.
5. Load your digest on adjacent lanes of the agarose gel as follows:
Lane 1: Leave this lane blank
Lane 2: 5μl DNA size marker (1 kb ladder)
Lane 3: 5μl uncut sample
Lane 4: Leave this lane blank
Lane 5: 30μl of your digested sample

6. Allow the gels to run at 100V until the bromophenol blue tracking dye is approximately three-quarter way down the length of your gel.
Whilst your gel is running, draw out a diagram of how your gel picture will look if your digestion is i) partial and ii) complete

7. Stick your photograph in your notebook. Label the lanes and write a brief account what the result showed. Have you achieved a complete digest? If not, how would you modify the experiment to obtain a complete digest?

Experiment 2.3: Ligate the gfuvpDNA into the pET28c expression vector
You will be provided with a pre-measured aliquot (50ng) of cut pET28c vector DNA for the ligation reaction. This has been digested with NdeI and HindIII, and incubated in the presence of Alkaline Phosphatase, Calf Intestinal (CIP) and cleaned up using the QIAquick PCR purification kit.

Note: your final total ligation volume should be 10µl. You will require 8:1 molar ratio of insert to vector [vector is 5.4 kb and GFP insert is 0.7kb]. Using the formula to calculate insert:vector molar ratio (above) you will need approximately 50ng of gfp insert DNA.

1. Calculate how many µl of the provided gfp insert DNA is necessary to give you 50ng. This is X in step 2.
2. Set up your ligation reaction as follows in a PCR tube (Note: not the standard small 0.5ml tubes) 1µl pET28c vector (50ng)
Xµlgfpinsert (50ng)
2µl of 5x T4 DNA ligase buffer water to 9µl
1µl T4DNA ligase (1 U/µl)

10µl final volume

3. Mix your ligation reaction by carefully by pipetting up and down 2-3 times.
4. Set up a negative control exactly as above but replace the gfp insert with sterile water.
5. Place the ligation reactions into the PCR machine that is set at 16oC. Ensure you have labelled your tube clearly.
6. Note: The lab technicians will do this part for you. Your ligation reactions will be incubated at 16oC overnight. The next day, the ligation reactions are removed and store at 4oC until the start of the next class

PRE-PRACTICAL QUESTIONS: PRACTICAL 2

Questions relating to Experiment 2.3 - Ligation
1. What is the purpose of incubating the pET28c vector in the presence of Alkaline Phosphatase, Calf Intestinal (CIP) prior to ligation?

2. In this experiment you will be attempting to ligate your gfpuv insert DNA into the expression vector pET28c. List the key features of a plasmid used for the expression of proteins (see Figure 1 of the vector maps in this workbook).

3. How do you know that the gfpuv coding region will be inserted in the correct orientation so that the protein can be expressed?

4. What is the purpose of the kanamycin resistance gene in the pET28c expression vector?

5. Make a drawing of the ligation products that can occur in your ligation tubes.

Possible ligation products in vector + insert tube (ligation-experimental)

Possible ligation products in vector only tube (ligation-control)