Assignment Task:
TASK:
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Aim and Learning objectives
This assessment provides an opportunity to utilise what you have learned in the course so far about experimental design. You will need to design three experiments that correctly address the hypotheses shown in the template on page 4 of this document. By successfully completing this assessment you will demonstrate the ability to:
Select appropriate techniques to address given hypotheses
Design properly controlled experiments and explain how they address the given hypotheses
Scenario and Assessment Notes
37 patients in their early 20’s have presented with symptoms described by medical professionals as “sudden onset dementia” The timing and pattern of presentation is consistent with a localised outbreak of disease, suggesting either a chemical or infectious cause rather than a common genetic defect. After extensive testing, no common chemical causes were able to be identified, suggesting an infectious cause for the disease. Concurrent epidemiological investigations found that all the patients attended a university barbeque for Biomedical science students several days before the outbreak. Further to this, it was discovered the only common food all of the patients had eaten was a beetroot salad. Bacteriological testing discovered a previously uncharacterised bacteria in stool samples from all of the affected individuals.
Before you start thinking about experiments, you should consider what information you can extract from the scenario. For example, if the beetroot salad was in fact the source of the bacteria, then how did the patients get infected? It would have to be self-inoculation by ingestion. If the ingested bacteria were in fact the ultimate cause of the observed dementia like symptoms, then you should be asking “How did a suspected gut pathogen somehow affect the brain?” This type of thinking is crucial to good experimental design; you always need to be considering the implications of the information you have available, and how that information can suggest interesting areas to research. Remember that this is an artificial scenario, and we don’t have or need to know every single step in the process between infection and disease. In fact, you need to make some assumptions (e.g. you have access to a specific reagent or mutant strain) in order to properly draft your experiments. The hypotheses you are basing your designed experiments on have been chosen as they highlight important steps in what might happen in real life, and they lend themselves to some key experimental techniques that are common in most biomedical research. For example, in the first hypothesis (see the last page of this document) you could consider making a mutant in the either the bacteria or the host cells to investigate the hypothesised binding activity.
Exemplar for the hypothesis “Bacterial Toxin X binds to T lymphocytes”Experimental Design (240 words, inclusive of in-text references)
To test the hypothesis “Bacterial Toxin X binds T-lymphocytes”, a flow cytometry experiment will be performed. First, a culture of mouse T-lymphocytes will be grown in AIM V™ Media (ThermoFisher Scientific, 2020) and split into 6 separate samples. Each sample will receive buffer solution with or without added toxin. Samples 1 & 3 will receive toxin buffer alone (negative control samples). Sample 2 will receive 1g/mL of purified Toxic shock syndrome toxin-1 (TSST; in toxin buffer), which is known to bind to T-lymphocytes (technique control). Samples 4, 5 and 6 will receive 0.1g/mL, 1g/mL and 10g/mL of purified Toxin X (in toxin buffer) respectively. As the binding affinity of toxin X for T-cells is unknown, this range of concentrations should allow for toxin binding detection where the binding affinity is higher or lower than that observed for TSST. Following toxin binding the T-lymphocytes will be washed (with AIM V™ Media) and incubated with either mouse anti-TSST (for cultures 1 & 2) or mouse anti-toxin X (for cultures 3-6). After incubation, all cultures will be washed to remove unbound primary antibody, then incubated with secondary antibody (fluorescently tagged sheep anti-mouse immunoglobulin). Following a final wash to remove unbound secondary antibody, all samples will be fixed with 3% paraformaldehyde (Sigma-Aldrich; 2014). After fixing the samples will be analysed using a flow cytometer to determine both the proportion of cells that have bound Toxin X, and the relative amount of Toxin X binding.
Conclusion (66 words)
The buffer-only negative controls establish the background autofluorescence in the presence of each primary antibody; values above this level in the experimental samples can be correctly attributed to the binding of toxin to the cells rather than to binding of antibody directly to the cells. The TT control ensures the technique is functioning as expected, although it does not control directly for the anti-Toxin X antibody.
Exemplar notes
This exemplar contains the level of detail expected. You DO NOT need to discuss statistical analysis; for the purposes of this assessment it is assumed each experiment has a statistically relevant number of replicates. Nor should you describe how individual reagents were generated; purified Toxin X was used in the exemplar, but nothing was said about how it was obtained, since that relates to preparatory work not covered in this assessment. Providing specific details about aspects such as reagent concentrations, times for incubation, volumes, reagent suppliers etc. is somewhat flexible. For example, the exemplar describes toxin concentrations but not antibody concentrations. This should make sense – the antibody concentration would be fixed at a level appropriate for this particular assay (i.e. it would be kept the same across all the samples tested) and as such the actual concentration isn’t critical for the experimental design. Given that we don’t yet know whether toxin X binds, specifying a range of concentrations makes good sense from an experimental design perspective – if you selected a single concentration, a lack of fluorescence might be due to the concentration being too low, rather than because it doesn’t actually bind to the T-lymphocytes.
You are not expected to go into excruciating detail for every single element of the experiment – this would take far too many words! Inclusion of some specific details adds a level of authenticity to your experimental design i.e. it shows that you have bothered to properly research how these experiments might be done! Keep your focus on the design aspect – getting the controls correct and using an appropriate technique to address each hypothesis is far more important than specifying what supplier a particular buffer came from. Use the exemplar as a guide and make sure you reference correctly!
Marking rubric (read this page carefully! Delete this page before submission)
Max mark Your Mark Grade Fail
(F) Fail
(F) Pass
(P) Credit
(C) Distinction
(D) High Distinction
(HD)
type=grid
ninc=1
ndpm=1
ndpt=1 0% to 39% 40 to 49% 50% to 64% 65% to 74% 75% to 84% 85% to 100%
Experimental design 1 (clearly explains the process for addressing hypothesis #1) 20 Critical details are missing, the design as stated does not address the hypothesis. Critical details are incorrect, indicating a lack of understanding. Experimental plan adequately addresses the hypothesis, but contains some errors. The experimental plan clearly addresses the hypothesis, with few or no errors.
Experimental design 2 (clearly explains the process for addressing hypothesis #2) 20 Critical details are missing, the design as stated does not address the hypothesis. Critical details are incorrect, indicating a lack of understanding. Experimental plan adequately addresses the hypothesis, but contains some errors. The experimental plan clearly addresses the hypothesis, with few or no errors.
Experimental design 3 (clearly explains the process for addressing hypothesis #3) 20 Critical details are missing, the design as stated does not address the hypothesis. Critical details are incorrect, indicating a lack of understanding. Experimental plan adequately addresses the hypothesis, but contains some errors. The experimental plan clearly addresses the hypothesis, with few or no errors.
Conclusions
(Clearly indicates how the experiments address the hypotheses) 30 The expected experimental outcomes are not described. The expected experimental outcomes are misinterpreted. The expected experimental outcomes are discussed, but some details are not properly addressed. All expected experimental outcomes are clearly and succinctly discussed.
Clarity (Grammar, Spelling and References) 10 Not understandable due to excessive grammar and/or spelling mistakes
No references are included Many grammar and/or spelling mistakes, making the expt. design difficult to follow.
Reference list is poorly formatted and incomplete Some grammar and/or spelling mistakes which slightly compromise the expt. design. Only minor grammatical errors which do not compromise the expt. design. Includes a complete and well formatted reference list
Penalties (late or over word limit) Comments Total 100 Percentage 100% Relative Semester Mark (as %) 35 Grade Assessment #3 – Name:
Word LIMIT = 1000 words
Total = 100 marks (35% of the semester total)
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Delete ALL red text, replace it with your own work (ensuring your submission uses BLACK font).
Experimental Design (~250 words/experiment, ~750 words total)
Based on your understanding of controls and experimental design, you need to design one experiment for each of the following three hypotheses.
The pathogen-secreted effector molecule is secreted after the pathogen binds to (colonises) gastrointestinal epithelial cells
The pathogen-secreted effector molecule localises in the cytoplasm of neurons
Expression of the mouse gene xyzA (which is already known to induce dementia-like symptoms when overexpressed) is upregulated in neurons exposed to the pathogen-secreted effector molecule.
Make sure you describe the required control samples – see the exemplar for the level of detail expected.
Conclusions (~80 words/experiment, ~240 words total)
This section summarises how your experimental design leads to an unambiguous answer for each hypothesis, with particular focus on how the controls allow for only one conclusion to be drawn.
References (Not included in the word count)
List any references used here.
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