Lab report guidelines

Bioprospecting for antimicrobials MBB265

Key format guidelines

• Include your: name, U Card number and the date (also identify    your colleagues in your group) 
• Summary/Abstract (50% A4) 
• Conclusion and future suggestions (50% A4) 
• Detailed diagram of protocol and results (A4 page) 
• Submission date Monday 15^th May (before 16.00)

The Abstract should capture the whole experiment, background, experimental, results and conclusion, in general terms. You may refer to your figure (although this isn’t usual in abstracts, it is fine here) [25% marks]

The Conclusion should summarise your key finding(s) and suggest further work or ways in which you might overcome any failures in the experiment [25% marks]

The Diagram can be hand drawn, may contain photographs, but should capture the essential stages of the work from start to finish. Importantly, it must be accompanied by a detailed legend and labels as appropriate [50% marks]

The sample figure below has a detailed legend, but it also needs a title?

Legend

Microcin C induces persistence in growing Escherichia coli cultures.
A. Growth curves of MG1655 (wt) strain after McC treatment. An overnight culture was diluted 100‐fold in LB and growth was allowed to continue at 37°C. When OD600 reached 0.5 the indicated concentrations of McC were added to culture aliquots and further growth was monitored by following OD600 at indicated time points.
B. As in А, but showing the number of colony forming units (CFUs) on LB agar plates at various time points after the McC addition.
C. An example of killing curves obtained after ciprofloxacin (Cfx) treatment of MG1655 (wt) culture with or without McC (1.5 μM). Cultures were grown as in A. After 30 min incubation with McC, 1 mg/l Cfx was added. The incubation was continued and culture aliquots were removed at various time points followed by CFUs determination. Mean values and standard deviation obtained from three independent experiments are shown.
D. For each killing curve obtained with or without McC, percentage of surviving cells (for details see Experimental Procedures) after 4‐h incubation in the presence of Cfx (see panel C) was calculated. Error bars show standard deviations of mean values of at least 3 independent experiments.

A couple of ideas for figures are given below 

Coconuts

The current level 2 class in MBB (MBB265) has become known locally as the coconut lab, mainly because we use coconuts as one of the sources of antimicrobial, natural products. As we approach the end of the experiment, I thought I would make a few observations about the highlights, the unexpected events. However, my main reason is to provide some additional support for the students who are about to collect their results and write their lab reports. The objectives of the coconut lab are several-fold. At one level, it is intended to bring together a number of skills that have formed part of the undergraduate experimental labs at Sheffield in Molecular Biology and Biotechnology, such as Microbiology and Biochemistry with some generic analysis methods thrown in. However the most important reason for the lab classes is to give students their first exposure to running an experimental research project. The instructions provided to students during the the first session are simply:

• Brainstorm ideas
• Organise and prioritise your ideas
• Develop an experimental plan
• Assemble reagents and equipment
• Begin

The Learning Objectives The first session, as you might imagine, was a little animated, students were coming to terms with the general responses to questions they would ask of the demonstrators: I don't know, what do you think? But everyone got used to it eventually and the questions were soon framed more thoughtfully. I was planning on using 6 discs on my plates, is this too many? Or, would you use distillation to separate aqueous and solvent phases, or centrifugation? It was clear that the ability to trouble shoot and make decisions had come on leaps and bounds and, while some students would clearly prefer a more structured lab class experience, with standardised protocols, this is unlikely to be closer to the kind of experimental work they will encounter in a PhD or commercial laboratory setting. 

The second aspect of the lab class that is different to regular classes, is that any failure to plan appropriately (i.e. make up solutions in advance or inoculate cell cultures in a timely way etc.) is met with a shrug of the shoulders and a suggestion to re-plan in a less ambitious way, or try and find a suitable replacement sample from a colleague. Again, placing the responsibility on the students' shoulders. And I say students, since this is a small group project and so poor planning and organisation should be minimised, providing the team share the responsibilities evenly.

The Science In addition to the general lab skills and research experience, the project has a relevance to understanding simple drug discovery strategies, in particular the search for new antimicrobial compounds. And in particular, an appreciation of the world of Natural Product Chemistry and Bioprospecting. The students are expected to develop an experimental strategy to establish whether extracts from a range of fruits, vegetables, roots etc., are capable of inhibiting the growth of one or more classic strains of bacteria: Proteus, Staphylococcus, E.coli and Pseudomonas.

Materials and Methods The students are asked to draw on their knowledge and lab skills to devise natural product extraction and testing protocols. They must choose the source material based on availability in the lab and then devise a way of making extracts and preparing them alongside a set of their own controls, in order to collect any evidence of growth inhibition. Students across the class may choose to prepare extracts from any or all parts of the plants. They may choose to look at aqueous or solvent based protocols and are free to use separation methods of their choosing, combined with ion exchange enrichment should they wish.

Data collection and analysis The impact of the extracts on growth may be monitored on plates, with or without top-agar or in broth culture. Both methods are satisfactory, the choice method(s) for plotting the data, to obtain a quantitative measure of growth inhibition (compared with common antibiotics), is at the students' discretion and I am looking for creativity in presentation of data as well, of course as critical evaluation. (I will add a supplement after I have looked through the lab reports). Communication of the results and design of follow up experiments is a critical part of the report. It is absolutely fine to discuss mistakes made, controls omitted: as long as lessons are learnt!


A final note on writing The communication of Scientific work, or indeed any form of evidential document (legal, historical etc) requires a high standard of English and precision: clarity of explanation and a careful avoidance of ambiguity, combined with a slightly formal style, is the "Gold Standard" here. A creative and attractive style of Science writing is also something you should strive for, but it is the icing on the cake and is often reserved for "reviews" or popular articles.

Finally, the story so far....The pattern of response to this kind of class is pretty standard. Students are generally a little anxious at first, but most embrace the freedom and the opportunity to follow their own ideas. The usual problems of inadequate labelling, mis-calculating concentrations crop up, but this is usually "contained" by the team approach. Estimating volumes of media for plating, and anticipating problems such as the premature setting of top agar etc and a general, or the need for a starter culture first thing in the morning are all apparent. However, I have been pleased with the planning and timetabling awareness: a key part of time management. 

Let's see what the results look like.... 

pH, folding and catalysis

The recent level one class (MBB165) on the influence of temperature and pH on the enzymatic reaction catalysed by aryl sulfatase made me think about explanations of classic "physical chemistry" effects on proteins in general. School Biology and Chemistry classes introduce students to the idea of pH and temperature optima for enzymes. In the case of temperature, explanations are pretty straight forward: as the temperature of a reaction increases, so too does the rate. You will recall that the reason for this in a typical uncatalysed reaction is not simply an increase in the number of molecular collisions, but a shift in the proportion of molecules that have crossed the "activation energy" threshold. Hence there is an approximate doubling of reaction rate for every 10 degree increase in temperature. However, when an enzyme is present, the increase is a consequence )or function) not only of the activation effect, but is also a function of the intrinsic thermostability of the enzyme. So, enzymes exhibit temperature optima as a consequence of reaction kinetics and protein stability factors. 

How might you design experiments to dissect out these two components?

Can you think of an important enzyme catalysed reaction (used by thousands of labs every day) which relies heavily on thermal control?

The pH dependence of a reaction turns out to be a little more complex and you need to first consider the pH dependence of the side chains of the amino acids found in proteins. I wont discuss it here, but the same would be true for the bases found in RNA, in the case of catalytic RNA molecules, or ribozymes. That's for another time.

Amino acids and their pKas. All of the common amino acids except glycine are chiral (have a handedness). The major form found in Nature are the S-amino acid, or L-amino acids . [The mirror image of each amino acid can be found in Nature, although they are less common.] In the Table below, the third, fourth and fifth columns give pKa values of groups: -NH₃ refers to the protonated alpha-amino group, the CO₂H refers to the carboxylic acid group on the alpha-carbon, and the side chain is only relevant for a few amino acids. And this is a very important point. The impact of pH on protein structure and function is therefore limited to the acidic class (Glu and Asp), the basic pair; Lys and Arg and importantly, the amino acid histidine which has a "special" place in proteins, since its pKa lies in the neutral or physiological range. In all cases below, pKas refer to the free amino acid, and not the amino acid incorporated into a polypeptide or protein chain. Usually, we assume that not much happens to the pK_a of the side chain on incorporation. While this is not strictly true, it is a good starting point, until the value can be measured independently. Polar side chains such as Ser, Thr, Tyr and Cys are important as Hydrogen bond acceptors, but in order to promote their reactivity, they generally need to be "influenced" by adjacent (in spatial terms) side chains, cofactors and in particular metal ions. Perhaps the best example of the key role of a Ser residue and the sphere of influence of the active site residues is given by the Serine Proteases.
 

Amino Acid	R	-NH3+	-CO2H	Side chain	pI
Glycine, Gly	-H	9.78	2.35		5.97
Alanine, Ala	-CH3	9.87	2.35		6.02
Valine, Val	-CH(CH3)2	9.74	2.29		5.97
Leucine, Leu	CH2CH(CH3)2	9.74	2.33		5.98
Isoleucine, Ile	CH(CH3)CH2CH3	9.76	2.32		6.02
Phenylalanine, Phe		9.31	2.20		5.48
Tryptophan, Trp		9.41	2.46		5.88
Tyrosine, Tyr		9.21	2.20	10.46	5.65
Histidine, His		9.33	1.80	6.04*	7.58
Serine, Ser	CH2OH	9.21	2.19		5.68
Threonine, Thr	CH(CH3)-OH	9.10	2.09		6.53
Methionine, Met	CH2CH2SCH3	9.28	2.13		5.75
Cysteine, Cys	CH2SH	10.70	1.92	8.37	5.14
Aspartic Acid, Asp	CH2CO2H	9.90	1.99	3.90	2.87
Glutamic Acid, Glu	CH2CH2CO2H	9.47	2.10	4.07	3.22
Asparagine, Asn	CH2CONH2	8.72	2.14		5.41
Glutamine, Gln	CH2CH2CONH2	9.13	2.17		5.65
Lysine, Lys	(CH2)4NH2	9.06	2.16	10.54*	9.74
Arginine, Arg		8.99	1.82	12.48*	10.76
Proline, Pro		10.64	1.95		6.10

*Refers to the conjugate acid.

In order to explain the pH dependence of enzymes we need to two major factors: (i) folding and stability and (ii) orientation and reactivity of active site side chains. These topics will be covered in great detail during your degree, but here is a taster of things to come.

Protein folding and stability is determined by well established principles of Physical Chemistry combined with the unique chemistry of biological polypeptides. As Anfinsen has discussed: the primary structure of a protein carries sufficient information to direct its folding into a uniquely active conformation. [There are some emerging caveats to this principle, but they can come later]. There are some difficulties encountered in the experimental investigation of protein folding and so despite Anfinsen's insight and choice of the "well-behaved" nucleases, detailed investigations of protein folding really became possible with the advent of high resolution NMR spectrometry. The second barrier to understanding the mechanism of protein folding has become known as Levinthal's paradox. In short, for a given polypeptide chain comprising say 200 amino acid monomer units, each monomer unit has the freedom to sample many conformations (rotation about bonds etc), and the time taken for such a polypeptide to sample all possible structures is incompatible with "biological time". Clearly, the cell has a solution and in level 3 you will come across lowest free energy states and the "folding funnel" model for protein folding. Let's simplify things here and consider the following stages of folding of a polypeptide chain emerging from the ribosome or a round bottom flask in the lab. First, the hydrophobic effect drives the polypeptide chain into several more compact forms, the acquisition of secondary structure elements often then leads to the formation of a metastable state and finally, the lowest free energy form of the functional protein is attained, usually in less than 1ms. I think it is pretty clear that whilst many secondary structure interactions involve main chain hydrogen bond donors and acceptors, the later stage tuning and stabilisation of the final structure involves side chain interactions. As a consequence, the potential for a pH influence becomes inevitable.The pH of the solvent can influence the properties of the active site of an enzymes by perturbing the ionisation of key residues. As you will have noticed, the pH profile of an enzyme is often (but not always) bell shaped. The peak of the curve represents the optimum pH for activity, whilst the descending "shoulders" tend to reflect the combined influence of pH on protein stability and activity:at extremes of pH, most proteins tend to denature. An interesting couple of examples to consider from the literature are the gastric protease pepsin and the Krebs Cycle enzyme fumarase. Pepsin is a proteolytic enzyme that is usually found at acidic pHs: interestingly as the pH of the solvent is raised from 5 to 7, pepsin begins to denature. Not surprisingly, it is an acid protease and aspartic acid is found at its active site.

Can you think of an aspartic protease that made the headlines as a potential drug target?

Fumarase was characterised in a lovely early pH study by Frieden and Alberty: two well known enzymologists. In a paper published over 50 years ago, they demonstrated the insight that could be obtained from a careful analysis of the pH dependence of an enzymatic reaction. Such studies underpin the broad view that ionisation of two or more key residues at the active site of an enzyme, is responsible for the pH profile. If we look at the active site of aryl sulfatases, not the one from the snail, Helix pomatia (the source used in the lab classes),but from a related enzyme found in bacteria (for which there is a very high resolution structure), it is immediately clear that it is stuffed full of ionisable side chains: see below.

The other interesting feature is the calcium ion at the centre. Can you think of a plausible mechanism for how such a clustering of residues could promote the removal of the sulphate group from the aromatic ring?

In summary, pH has the potential to influence protein function through structural perturbations and by modifying the degree of ionisation of key active site side chains. As you can probably tell, methods of analysis are needed that identify the contribution of all amino acids in a protein during catalysis. Such methods will come, but for now we will continue to make educated guesses to supplement experimental data.