BISC209/S11: Assignment 209 Lab5

Materials and Methods

Due at the beginning of Lab 6.
How to Write a Materials and Methods Section
It can be a daunting task to write a single paper on several months of experiments, particularly when those experiments result in a huge amount of disparate data. Turning an entire semester of lab work into a Materials and Methods section for your final paper can be particularly overwhelming, but it need not be. It is less so if you have kept a good lab notebook that contains all the information necessary to explain how to make or purchase reagents and clearly explains how you used these materials to accomplish your small and large goals.

It is also much easier if you think of your semester's work as a whole project that involved many experiments to gather specific evidence for aspects of the investigation. You must organize everything by goal and what you did to accomplish answering each of our experimental questions: How many microbes are in our soil community? Who are they and how are they related to each other? How do they compete and co-operate to function effectively as a community? Organization of your M&M section by goal and only as a subsection by independent tools is MUCH better than trying to write a chronological description of everything you did each day. A time-course description of the semester's lab work is NOT the way to organize and report the materials and methods applied to this project. The focus of a good M&M section is on how each of the stated goals was accomplished. The wiki protocols tell you exactly what to do in what order, recognizing that you are new to most of these tools and materials. The detail in these protocols is far beyond anything needed in M&M and the focus is wrong. It would make your M&M section unreadable and unintelligible if you were to include all the wiki's trivial directives about what to do to tubes and plates at each step of every protocol. The wiki also lacks, in these detailed chronological lab day descriptions, lots of crucial information that you need in M&M. The wiki, largely, ignores all the behind the scenes work required to make or acquire the materials necessary to accomplish the work described. The ingredients and concentration information needed to make media,etc. or the information required to order reagents and use them properly must be included in a M&M section. Fortunately, if all that information has been previously published or can be found on a kit or reagent suppliers web-site, you need not repeat it-- as long as you include sufficient information in your M&M for a reader to find the supplier or manufacture's product information or you have the full citation to the paper or methods manual that describes how to make all the materials and to perform the testing. You MAY NOT cite the wiki for any information.

To help you with this assignment we have provided an organization scheme for your M&M section and have written a few sample sections for you. The best way to learn how to write M&M properly is to look at published papers that use similar tools and see how they described the use of those tools. If you find a reference to a publication that may describe a protocol you performed, you must look up that reference and be sure they did the procedure the way you did and, if there are slight modifications, describe them.

How to Hierachially Organize the M&M section of this project?
First describe any procedures common to the three basic divisions: Each should be separately titled. If you did them more than once, there is no need to mention that. Separately title each protocol, stressing the goal rather than the tool in the title. Order them by deciding which goal must be accomplished first. (Note that you do NOT have to write up protocols that you have not performed yet. This list is to help you know what to include in your final M&M section.)
Acquiring a Soil Sample for Bacterial Community Identification and Assessment
Making a Soil Extract

After describing general protocols common to different lines of our investigation, create categories that describe general lines of investigation that we are using to provide evidence for abundance, diversity, co-operation and competition in a soil community: enumeration, identification and establishing phylogenetic relationships, cooperation and competition

1. Enumeration of Soil Community microorganisms
   1. By Culture-Independent direct count of nucleic-acid aggregates (nuclei or bacterial chromosome)using Dapi
   2. By Culture-Dependent plate count
2. Culture-Independent Identification of Bacteria in a Soil Community by 16s rRNA Gene Sequencing of whole soil genomic DNA(notice goal is before tool)
   1. Isolation of genomic DNA from soil
   2. Amplification of 16s rDNA by polymerase chain reaction. This protocol should include the adjustment of DNA concentration, the pcr product "cleanup" and the assessment of success by agarose gel electrophoresis). The final goal of this protocol is to end up with lots of copies of double-stranded DNA amplified from the 16s rRNA gene template found in as many as possible of the bacteria in your soil community.
   3. Separating the 16s rRNA genes of bacteria in a soil community. This protocol should include ligating the 16s rDNA into different copies of a plasmid vector; transforming chemically competent E. coli with the vector plasmids with 16sRNA gene inserts; selecting for appropriate isolated transformants; preparing glycerol stocks of successful, cultured, separated transformants; DNA sequencing of 16s rRNA genes from clones (name the sequencing method, facility and location we will use)
3. Soil Microbial Co-operation & Competition
   1. Culture-Dependent Microbial Community Assessment
      1. Community Physiological profiling: Carbon source utilization: average metabolic response (AMR) and community metabolic diversity (CMD), carbon utilization patterns
      2. Culture-Dependent Assessment of Exoenzymes in the Community: Cellulase, Amylase, Phosphate Solubilization
   2. Examples of co-operation and competition in cultured bacterial isolates from a Soil Community
      1. Isolation of Methylotropic Denitifiying Bacteria & Nitrogen Cycling bacteria such as Azotobacter to Pure Culture
         1. Functional testing for role in the nitrogen cycle
      2. Isolation of Spore forming Bacteria to pure culture
         1. Antibiotic Production/Sensitivity Testing
      3. Mutualistic and Antagonistic Interaction Assessment
      4. Quorum Sensing Assessment

General tips:
Please look at the Methods sections of published scientific research papers (there are many in the References tool in Sakai) to see how the protocols in this wiki differ from Materials and Methods descriptions in a scientific paper. Notice that M&M can be very short, particularly if you have used a kit or a previously published protocol for a part or all of the experiment. If some part of your work is published elsewhere, such as in a journal article's methods section or in a kit manufacturer's website, you may reference the kit manufacturer or use a journal article citation to the previously published directions.

Remember that all separately sectioned protocols are independent; therefore, you can not assume your reader knows what soil sample or extract you mean unless you say, "soil sample obtained as described in Materials and Methods".

In order to write about how you accomplished your experimental goals succinctly and clearly, you must thoroughly understand what part of your goal is accomplished in each step. It does not require thorough understanding to follow the directions in the wiki and end up with a successful experiment; however, to write about your experiments in such a way that someone who is not part of this course can understand what you did and how it contributed to your overall goal... that's is not so easy. If you make sure that everytime you leave lab you understand how each part of each day's work fits into the project's goals, writing M&M, as well as the other parts of this paper, will be manageable.

Keep in mind that when you reference a reagent manufacturer's web site (such as the New England BioLab's website directions for using Phusion® High-Fidelity PCR Master Mix with HF Buffer-- the reagent we used to amplify our target DNA by pcr (found at: | http://www.neb.com/nebecomm/ManualFiles/manualF-531.pdf), the manufacturer does not include the sequences of the primers we used (since those are unique to your target DNA) nor the exact thermal cycler program (since that is tweaked for the length of the DNA section you want to copy and for the relative CG content of the template DNA). Therefore, you must give more than a brief citation and web address. In the case of the pcr amplification using a commercial master mix, you must be specific about the template DNA, the primer information for amplifying 16s rDNA, and give the exact thermal cycler program you used.

Protocols are instructions to “do this, then that” in an exact, detailed time-course set of instructions for YOU to follow. Materials in Methods should be none of that; rather, the M&M is divided into discrete sections (sometimes with subsections) that each begin with a title that states the smaller goal that will be accomplished and then explains how one accomplishes that goal from the starting material to the end point that is described in the title. Stress the goal and not the tool! For example, do not use the title “polymerase chain reaction” for a section in your M&M since that only describes the tool and says nothing about the context or goal. A better title would be, “Amplification of 16s rDNA by polymerase chain reaction (pcr)”.

Although it is important for your audience (who knows nothing, necessarily, about soil or bacteria) to be able to understand how the goal of each part of the experiment was reached, don’t spend time here explaining a lot of theory (omit why DNA sticks to a silica gel or is precipitated in ethanol and solubilized in water or water based buffers). M&M is a “how to do it” section; it is not so much a “why does it work this way” section.

Effective Concentration: You should use concentration rather than volume or weight terms whenever possible. Examples are % (vol/vol or wt/vol), or wt/vol terms such as mg/ml, or molarity. For example, instead of saying, “we combined 1gm of soil with 100 ml of water”, say, “A 1% soil extract was prepared in sterile water”. You can assume your reader is a savvy enough laboratorian so that if he/she wants to follow this protocol that means that he/she must weigh out 1gram of soil and dilute it in a total volume of 100 ml of sterile water. Note that you have changed the focus away from what YOU DID to how the goal was achieved (making a soil extract) and you haven’t given details that any laboratorian should know (like weighing, mixing, etc).

Sometimes it is not possible to give concentration; therefore, you must use volume or ratios of volumes. For example, when you resuspend cells in liquid medium, your reader needs to know approximately how concentrated the cells should be. If you make them too dilute or too concentrated it will negatively affect the goal, but sometimes (such as in our transformation) you don’t know the concentration. Volume is all you can do there.

Avoid using the words “tube” or “plate" Tubes are just pieces of glass, and plates are empty pieces of plastic; neither is an accurate way to describe what is happening in a culture or a step in an experiment. If you find yourself mentioning tubes or plates, stop and ask yourself, “what’s in the tube or plate?” and rephrase with a brief description of only the crucial info.

Rather than explaining what you did to your “tubes” or “plates”, you should change the focus to how the goal was accomplished. One description of isolation of Hyphomicrobia to pure culture might read like this:
EXAMPLE:
Isolation of Denitrifying Methylotrophic Bacteria from Soil
Denitrifying methyloptrophic bacteria were initially enriched and selected from an approximately 2% (wt/vol) soil sample (obtained as described previously in M&M) in liquid denitrifying methylotrophic medium with methanol (DMMM: 1% Freshwater Base [FWB: 10% NaCl, 4% MgCl2*6H20; 1% CaCl2*2H2O; 2% KH2PO4 (acidic); 5% KCl]; 0.02M 3-N-morpholino propanesulfonic acid (MOPS C7H15NO4S pH 7.2), 0.2mM Na2SO4; 0.15mM K3PO4 pH 7.2; 5.0 mM NH4Cl, 0.5% KNO3; pH 7); 1% vitamin mix (Sigma product #M7150 -Murashige and Skoog Vitamin Powder); 0.25% methanol) at 30C in anaerobic conditions achieved by displacing as much air as possible with medium in a tightly sealed culture tube. Observation of turbidity and bubbles of nitrogen gas were used to indicate when secondary enrichment/selection was appropriate. A ~2% subculture was made in liquid DMMM using the same culture conditions. Production of nitrogen gas was used to determine when to subculture the secondary enrichment/selection to solid DMM medium (DMMM without methanol +1.5% agar). Oxygen was reduced and methanol was available during culture by incubation in a closed jar with a methanol gas enriched atmosphere and incubated at 30C. Serial streaking for isolation on DMM solid medium on sequential subcultures over several weeks produced isolated clear, glassy, drop-of-water colonies that were selected by subculture and isolation streaking. A pure culture of bacteria resulted, presumed to be a denitrifying methylotroph, proposed to be Hyphomicrobia. A pure culture of this isolate was maintained aerobically on solid medium: peptone yeast calcium maintenance for Hyphomicrobium (PYCM: 0.25% peptone; 0.05% yeast extract; 1 mM CaCl2; 2 mM MgSO4; 1% agar) at room temperature.

Notice some things I did and didn’t do in the example above. Your protocol tells you to add 0.5gm of soil to a screw capped tube of liquid medium and then add medium until it is very full. The M&M version I wrote tells you the approximate ratio of soil to medium (2%). Because there is probably about 25ml in the tube, when you have filled it completely, I'm approximating that 0.5g/25ml is x=100ml = 2%.
Although I told you in a previous section that you should NOT use the word “tube”, I just broke that rule. Why? Note that I didn’t say that I added soil to a “tube” or that I incubated the “tube”, which are the uses I want you to avoid. I need, however, in this situation to describe how I achieved a somewhat anaerobic environment without making the reader think I used a GasPak or anaerobic incubator since both of those alternatives are expensive and not necessary here.
Note that I didn’t give you all the ingredients for DMM. Can the reader make DMM from the information given? Yes, since I have given IN THE SAME SECTION the recipe for DMMM and told you that DMM is DMMM without methanol and with added agar of 1.5% (which would be 15g/L if you wanted to make that much).

Here is an example of a transformation protocol reworked as M&M. Note that this transformation was done in a completely different context. Every step isn’t exactly the same as our transformation. Nevertheless, if you compare the M&M version below to your lab wiki’s transformation protocol, you can get a feel for which information is essential and what can be omitted. You can also see what needs to be added so that an investigator can figure out how you accomplished getting plasmid DNA into E. coli and selecting for appropriate transformants.

Example:
Transformation of Reporter Strains of E. coli with pTC001
4ml of overnight culture of genetically modified reporter strains of Escherichia coli, 219B3-5 , designed to differentiate functional defects in λ repressor protein (see Table 1. for source & construct information), were prepared in Luria Bertoni broth (LB) containing 1% tryptone, 0.5% yeast extract, & 1% NaCl ). Cells were incubated at 37C with rotation. Intact cells were pelleted by centrifugation and made competent by exposure to 100mM CaCl2 at 40C for 1 hour. Competent cells were harvested by low speed centrifugation and resuspended in cold 100mM CaCl2 before transformation with isolated plasmid (pShrimp) DNA in a 20:1 volume ratio of competent cells to plasmid concentration (See Table 1 and M&M for plasmid construct and plasmid isolation procedure). Transformation mixes were incubated on ice for 10 minutes to allow uptake of plasmid DNA and then heat shocked at 42C for 90 seconds to facilitate transformation and to upregulate metabolism. The cells were diluted in LB broth in a 1:6 volume ratio of cells to LB and incubated for approximately an hour at 37C to facilitate expression of selection proteins in the transformants. An aliquot of cells predicted to yield between 30-300 colonies of transformations was spread onto slightly dehydrated LB agar (2%) solid medium containing 0.2mg/ml ampicillin. Cultures were incubated at 37C until visible colonies formed. Selection of transformants was accomplished on the basis of plasmid based ampicillin resistance. One well isolated colony of cells from each strain (219B3-5) expressing pShrimp genes, was inoculated into 4ml LB with 0.1mg/ml ampicillin and incubated overnight at 37C in order to harvest sufficient cells for quantitation of lambda repressor controlled expression of the reporter gene, lacZ.

Note that your transformation description must either describe or cite a description of the cloning vector and the strain of E. coli cells used and you MAY NOT cite the wiki anywhere in your paper. Since we used a kit, Invitrogen's Zero Blunt® TOPO® PCR Cloning Kit, you have the option of checking to see if that information and the directions for accomplishing the cloning and transformation is provided on the manufacturer’s web site and, if it is and that’s what we did, you can have a shortened section for the vector ligation and transformation section of M&M.

Don’t end a section of M&M until you make sure that the goal stated in your M&M section title has been clearly accomplished. Plating potential transformants onto LB+kan+Xgal is not the end. Your reader has no idea what happened next, and what's next is crucial information for understanding how you accomplished separation of your soil bacteria's 16srRNA genes. If you continued with, “Transformed cells expressing plasmid genes were selected on the basis of kanamycin resistance seen as colony formation on solid medium with kanamycin and cells containing vector plasmids with a soil bacterium 16s rRNA gene insert were detected as non-blue colonies on the basis of disruption of expression of LacZ and a ccdB gene that would prevent colony formation and/or a visible blue X-gal cleavage product in the colonies”; your reader would understand how you were able to reach your real goal: separation and harvesting of 16s rRNA genes from bacteria in your soil community.

Using a kit: When you use a kit, you often don’t have all the information that you need to explain to your reader how to make all the reagents because they are proprietary, so you do the next best thing; you include where to buy the kit and a link to the manufacturers website for the directions on how to use the kit to accomplish the goal. Kit M&M descriptions are simple and short; however, the section does need to start out with the goal and to include the starting material and ending material. For example: PCR products were removed of unincorporated dNPTs, primers, and other small fragments of DNA through use of Epoch BIoLabs GenCatch PCR CleanUp Kit (http://www.epochbiolabs.com/). This clean-up was accomplished by following the manufacturer’s directions.

Miscellaneous:
RPM’s are not universal but “g” force or rcf’s (relative centrifugal force) are terms that denote a centrifuge speed that can be achieved in any kind of centrifuge. Please use the universal terms, not the ones that only matter in a particular rotor in a particular centrifuge.

Use the full formal terms first time in each separately titled section. If there are acronyms that you want to use later, introduce them with the full term, ie. Polymerase chain reaction (pcr), nutrient agar (NA), Luria-Bertani (LB), etc.

There is an extensive handout on writing a Methods section in the Resources section of this wiki.