BISC220/S10: Mod 1 Lab 5

Writing the β-Galactosidase Scientific Paper
Your results will be described in a formal lab report that models scientific writing found in scientific journals. All parts of the report writing must be done individually. Each section is discussed in the order you should write the report. Title The title of your paper is what draws your reader in and catches their initial attention. DO NOT focus on the methods used. Instead focus on the CONCLUSION of the entire paper - what did you find out that a reader would find interesting! Materials & Methods When writing a scientific paper for publication, it is important to include sufficient information about the Materials & Methods so that other researchers could replicate your results. For these reports, the BISC 220 WIKI is NOT considered to be a “published” reference. Thus you should NOT cite the lab WIKI - or your lab instructor as a source. You should describe the components of the assay, the time of incubation, the range of substrate concentrations used, etc., but you should not include excessive detail, such as the level found in the actual lab manual protocol. It is difficult to decide how much detail is enough and when detail becomes excessive. It is not essential to supply the kind of tube, pipet, or incubation equipment you chose to use, since there are acceptable alternative ways to perform most general lab techniques. It is assumed that care is taken in performing all lab work so there is no need to include directions such as “carefully….” A less obvious example of inconsequential information is the specific dilutions of BSA that you used for the total protein assay. What the reader needs to know is the range of the BSA concentrations (0.1-1 mg/ml) used to generate a standard curve for a dilution of the CE and PF unknowns (1:5) that was expected to give an Absorbance reading within the range of the standards. Volumes are rarely as important as calculating effective concentrations of each ingredient in a reagent during a reaction. For example, in your SDS-PAGE description it is not necessary to explain all the steps you used to dilute the CE and PF since the important thing is that the protein samples were diluted in Laemmli buffer to end up with 5 µg protein applied to each lane. Enough information to be able to make Laemmli buffer is also crucial (ingredients and concentrations). The rule of thumb for deciding what to include is to ask yourself, “Is this information crucial to the success of the experiment?” If the answer is “yes” then you should include such information. Incubation times, temperatures and concentrations is critical to the success of the protocol. Other less important information can be omitted, but only if you cite a source that allows a reader to find it. Your materials and methods section should not be one long unbroken narrative, nor should it be composed of steps in a list, as in the lab manual. It should also NOT be a time-course description of everything that you did in lab over the last 4 weeks. For example, it is not important that the reader know that the cell pellets were frozen for a week. You could have continued the experiment that day or a month later. The timing was a personal choice and, therefore, a detail acceptable to omit. The temperature at which the cells were frozen, however, is important since we were assaying temperature sensitive enzyme activity, and activity is best preserved at very cold temperature. The storage temperature should be included since it might make a difference to the success of the experiment. Materials and Methods should be divided into discrete parts with headings that tell the reader what was accomplished by the procedures described under that heading. All protocols should be arranged in a clear progression that may or may not be the order in which you actually performed the experiments. Materials and Methods should be written in the past tense since you already completed this work. Passive voice is generally preferable to 1st person active voice in this section because it keeps the focus on the experiment and away from describing your lab day. Here is an example of how one part of the M&M section for your β-gal lab report might be written: ''Induction of 6xHis-β-galactosidase A 200-ml culture of BL21(pET-14b) Escherischia coli cells grown to a density of 0.5-0.7 A600nm in Luria broth (LB: 1% tryptone, 0.5% yeast extract,1% NaCl), minus a 1-ml aliquot, was induced with 1.0 mM isopropyl-beta-D-thiogalactoside (IPTG) for 1hour at 37°C. A post-induction aliquot of 1ml of cells was also reserved. The pre and post-IPTG induced aliquots were frozen at –70°C and saved for SDS-PAGE analysis. The remaining induced cells were collected by centrifugation and the pellet was frozen at –70°C.'' ''Purification of 6xHis-β-galactosidase The induced BL21(pET-14b) cells prepared according to “Induction of 6xHis-β-galactosidase” protocol described previously, were lysed and the protein extracted and purified using a B-PER 6xHIS Spin Purification kit (Pierce, Inc.) following manufacturer’s directions. After the cells were lysed through mechanical and chemical means in Bacterial Extraction Reagent™, the cell debris was pelleted by centrifugation. An aliquot of clarified lysate sufficient to perform total protein analysis, specific activity assays and SDS-PAGE visualization was saved as a Crude Extract (CE) sample. The remaining volume of Crude Extract was incubated with a 1:6 ratio of extract to nickel-chelated agarose suspension™ for affinity chromatography purification. The agarose was washed with manufacturer-provided wash buffers™, and the bound protein was eluted with imidazole containing Elution buffer™ to yield the Purified Fraction (PF). See the Pierce, Inc. web site (http://www.piercenet.com/) for more information concerning the chemical composition of the reagents used in the B-Per 6x His Spin Purification kit and the manufacturer’s instructions for use.'' Results The results section must have 2 parts: figures and/or tables of your processed data and a brief text description that explains how the data were generated and that points out what is most significant about those data. Raw data, e.g. tables of absorbance measurements, should not be included in the results section. Data such as sample calculations, formulas, and standard curves should be included in an appendix and referred to in the body of the paper and/or appropriate figure legend. The following tables and figures should be included in the results section of this paper: The figures should have properly formatted legends. Figure legends go below the figure and table legends go above the table. Legends for the reports in this course should not include a summary of the results or an interpretation of the data. The exception to that directive is the figure title which often is a statement of the main conclusion from the data presented in the figure. The legend itself should describe briefly, but clearly, the experiment that produced the data. It should not repeat all the details of the M&M section or the evaluation of the results found in the text. Please refer to the comments made about the legend of your RasMol figure and see your instructor for help if you are unsure about legend writing. If you use Excel to generate graphs as figures, note that Excel places, by default, a title at the top of the graph. Either delete it or move it to be the figure title just after the figure number. Please use your page break command to make sure that your complete figure or table with all of its legend is on the same page of your report. Figures and tables should be integrated into the results text. A figure or table should be inserted after its text description. The text description should refer to the table or figure by number. The text component of the results section should be structured around the data presented in Tables and Figures, but it should have a narrative flow. It should begin with an introduction of the purpose of the experiments and how the data were generated, but it should not completely repeat the Materials and Methods section. You should not start the section with a sentence like: “Figure one shows the Michaelis-Menten plot for β-galactosidase with and without the IPTG .” A beginning more helpful to your reader might be: A 6xHis-tagged form of β-galactosidase purified from E. coli extracts by nickel-chelate affinity chromatography was used to characterize the kinetic properties of the purified enzyme. Activity assays were performed using an appropriate range of concentrations of the substrate, ONPG, with and without the presence of the putative competitive inhibitor IPTG. Activity was determined by product formation/time/mg enzyme spectrophotometrically by measuring A420nm, the peak absorbance for the product of ONPG cleavage by β-galactosidase, ONP. Figure 1 shows the activity data in the form of a Michaelis-Menten plot. Next, point your reader to the data that are most important and make simple conclusions from those data. For example, in the part of the results text preceeding the Michaelis-Menten plot, you might point out (if this is what you found): The Vmax values for the reactions in the absence and presence of IPTG were similar, while the Km values were dissimilar (Fig 1). The inhibited reaction gave a higher Km value than the uninhibited. This pattern indicates that IPTG acted as a competitive inhibitor in this reaction. Note that this sentence shows the reader what she or he should notice in the data presentation and indicates why it is important, rather than just repeating the Km or Vmax numeric values. Save for the discussion the more explanatory parts of the data analysis (the connection with background material, the comparisons with data from other studies, the connections with data from other parts of this study and, most importantly, the mechanism competitive inhibition that gives this pattern of Vmax and Km. Discussion Your discussion is the most important part of the report and it must, like the data analysis in the results text, be structured around your findings (data). It should refer directly to those data by figure or table number as often as possible. Remember that you are writing this report for a reader who has general knowledge of biological laboratory methods and scientific vocabulary, but did not do this series of experiments and may or may not know anything about this topic. Write the discussion for a reader who does not know, necessarily, anything about your enzyme, ONPG or IPTG, or how to interpret Michaelis-Menten and Lineweaver-Burk plots. You must define or explain how the enzyme kinetics measurements are used, how you interpret what they mean and explain why the conclusions are significant. Include information about the structure of IPTG and ONPG that might be useful in supporting the conclusions made from the Lineweaver-Burk plot. Literature references must be used to support or clarify your findings, but they should not be the focus of the discussion. The discussion should begin with a brief reminder to the reader of the topic of the investigation. In this case, the general topic has 2 parts, the induction and purification of the protein, beta-galactosidase, and the examination of its catalytic activity in vitro. A non-natural substrate, ONPG, was used under saturating and non-saturating conditions and in the presence and absence of a compound that was predicted to be an inhibitor. Making and purifying protein preceded the assessment of activity so think carefully about the order in which you discuss the data. The discussion should then summarize the conclusions made in the results text in a logical progression. Since your purification success was determined by the calculations done after lab 2 and by the gel results in lab 4, the analysis of these data should occur early in the discussion even though you performed the gel electrophoresis last. Use the gel data to assess the success of the initial induction and the homogeneity of the protein and make a comparison with the specific activity and physical purity measurements. Point out whether the measurements support each other or seem contradictory. If contradictory, try to explain why. You calculated a molecular weight for β-galactosidase from your gel. Use the literature to find a reported weight for the holoenzyme. Explain any inconsistencies. The specific activity of β-galactosidase was measured in saturating and non-saturating concentrations of substrate, and in the presence and absence of an inhibitor that you hypothesized to cause a competitive type of inhibition. Use your Michaelis-Menten plot to illustrate points about enzyme kinetics in general and about β-galactosidase specifically. This would be a good place to make sure your reader understands the definition and signficance of Km and Vmax. Do not assume your reader understands Km or its relationship with substrate affinity. Remember not to speak hypothetically or in future tense about data you have already collected and analyzed. The Lineweaver-Burk plot should be used to help your reader understand the nature of IPTG as a competitive or non-competitive inhibitor. Use the literature to support or refute your conclusions. It’s ok to find and use a contradictory study if you include some conjecture about why the results might be contradictory. For example, if your Lineweaver-Burk plot seems to indicate that IPTG is a not a competitive inhibitor because the inhibited and uninhibited activity assays show very different Vmax values, and you believe your data unreliable because all previous studies confirm your hypothesis that IPTG acts competitively in this situation don’t make a conclusion in which you have no confidence. You might say: ''“Although in previous studies (Author, date and author, date) IPTG has been shown to be a competitive inhibitor of the catalytic function of beta-galactosidase, the Lineweaver-Burk plot (Figure 3) created from activity assays in the presence and absence of IPTG (Figure 2) in this study seems to indicate that IPTG does not compete with ONPG for the active site because the Vmax and Km comparisons show…. The activity measurements presented here should be repeated before conclusions as to the nature of this inhibitor can be confirmed as contradictory to previous findings.”'' If your data contradicts the literature but you feel your experiment and data are reliable then point out the contradiction and, if possible, hazard a brief guess as to what might be the cause. For example, ''Bigshot & Hotshot (2004) found IPTG to be a competitive inhibitor in a similar experiment using pure enzyme. Since the enzyme used in this study was only purified 1.8 fold over the crude cell extract and specific activity was only compared to their value of, it is possible that there were other interfering proteins, perhaps even an unknown non-competitive inhibitor affecting the reaction studied in this experiment. This experiment should be repeated using a more highly purified form of the enzyme with significantly greater specific activity before conclusions as to the nature of the competition can be formed.'' Use both your data and your literature findings in your explanations. It is important to know if your Km, Vmax, and Ki values agree with other studies. Check to see if your specific activity approaches the specific activity reported for the pure enzyme. If not, how might you explain this discrepancy? Please do not include a formal “sources of error” section in this report. If you feel that the reliability of your data is so uncertain that it affects your ability to make conclusions, as briefly as possible point that out and then move on to data that does allow you to form conclusions. For example, do not trash your data by saying, “the BSA standard curve generated to determine total protein concentration had an R2 value of 0.35 so we had to use another group’s curve”. If the standard curve you used to calculate your protein concentrations had an r2 value of ≥ 0.95, then the concentrations can be considered reliable. There is no need to confess to your reader that your own dilution making wasn’t great if you have confidence in the curve that you used.
 * 1) A table reporting the protein purification results. A good example is Table 1 in Lab 2
 * 2) The photo of your SDS-PAGE gel results as support for your induction & purification results.
 * 3) A Michaelis-Menten plot of velocity vs substrate concentration in the presence and absence of inhibitor, on one graph
 * 4) A Lineweaver-Burk plot of 1/v  vs. 1/[S] in the presence and absence of inhibitor, on one graph
 * 5) A table comparing your Vmax, Km, and Ki value(s) to values reported in another primary study (reference). Remember you can only compare values with the same units so you may have to do some conversions for the data in this table.

Always end with something positive rather than with all the things that went wrong. The discussion should conclude with a brief summary and include, if possible, the broader significance of the investigation. Introduction The introduction should be written last (except for the abstract), should be fairly brief and somewhat general, yet should direct the reader to the topic. It should introduce and give a bit of background information about the importance or significance of the topic so that the reader becomes interested enough to want to read the paper. The introduction gives general information necessary to understand the topic and the experimental questions. You can assume your reader knows basic scientific vocabulary so that you don’t need to explain what an enzyme is, but you should not assume that your reader knows what β-galactosidase is or does. You should also include, briefly, other information important to understanding this investigation. For example the structural similarity of lactose, ONPG and IPTG must be included somewhere, either here or in the discussion. Your reader must understand early in the paper what you are doing and why, but the introduction should not go into so much detail that the results and discussion sections are repetitive. You should not include background that is unrelated to this investigation. Explaining the lactose operon or including information about β-galactosidase activity in vivo is not appropriate. The introduction should also include a VERY general and BRIEF outline of what you will do to investigate the topic. For example, saying protein will be obtained by microbial induction and affinity purification is enough detail for that part of the experiment. The introduction should not include results or conclusions from your data and it should not be in as much detail or repeat the Discussion or the M&M sections. The introduction should not be more than 1-2 pages and should NOT start with “The purpose of this lab is…”. Do not write this paper as a lab exercise, but rather as a set of experiments performed to investigate this particular enzyme and to answer the experimental question (s) described. Abstract The abstract is the only part of the paper that is single-spaced. It should not be more than a paragraph, but it should include a bit of every section in the report. Therefore it is easiest to write it last. The abstract starts with the topic, describes the investigation, gives the most important SPECIFIC results (e.g. values of kinetic parameters determined in your investigation), the conclusions (e.g. type of inhibition observed for IPTG), and ends with the significance of the findings. To see how to write an effective abstract, please pay close attention to the abstracts in the articles you use as references from the published literature. References You must use adequate outside literature support. You have been given some suggested peer-reviewed journal article sources. Those articles are found in PDF files on the lab conference or in the E-Reserves Lab folder on the course conference. Those sources must be cited in proper format in the body of the report AND in list form in the reference section at the end. Sources not cited in the body of the paper are not listed in the reference section. Source material should not be quoted directly. Always paraphrase. You have been taught in your introductory biology courses two widely accepted models for reference format, numbering and alphabetical by author. For this course please use the later. The body of the paper should contain citations in the name/year format (Author, date) and at the end of the paper the reference list is alphabetized by author’s last name. Please use the CSE style name, year citation system. A web site with more information, particularly about how to cite electronic sources and sources other than journals is: http://www.lib.unc.edu/instruct/citations/cse/ Remember to cite all references. If your reference is an unpublished source, such as something you heard in lecture or from a lab intro please try first to find a published reference for that information. If you can’t find a published source, cite the source as a personal communication. In the body of the paper the citation might be, (Harris, per. comm.). You should not include personal communications in the list of references at the end. Appendix The results section generally includes only processed data. Your standard curves or the formulas and sample calculations used to form the data in the table and figures in the results section should be placed in an appendix so that your reader knows how the data in the tables and figures were generated. Note: To get a better feel for the proper style and content of the various sections of the lab report, you should note the way the published research papers are written. Be aware, however, that different journals have different stylistic requirements. You will not find one universally applicable way to write figure legends, M&M, or results and discussion sections.