Bacteria [Communications]

[donnahardy2]

Hi Trader,

Here are some answers to your questions.

1. I don't know how difficult it would be to construct a gfp-based biosensor for detecting AHL's. The E. coli you probably have available to use is available separately from the gfp plasmid, which is used to transform the bacterium. I don't know how you could use that to detect specific AHL's. Fortunately, I work with a group of molecular biologists, and I will consult with them and try to get a better answer for you. The list of materials you have included would be sufficient for growing E. coli. Do you have a reference for constructing a biosensor? I haven't found one yet, and this would be very helpful in answering this question.

2. Yes, the bioassay detects the presence of AHL’s; the TLC can be used to identify the length of the carbon chain and therefore the specific AHL. The two methods would require two different experiments and would provide complementary information, but would not be different variables. You are probably the expert at this point to check originality of the topic, but you are probably correct that this is a completely unique experiment.

3. Ethyl acetate is one of the solvents that are used to develop the TLC plates. One of the references I found (Shaw, et al, Proc Natl Acad Science, 94(12): 6036-6041, 1997, used a mixture of 60:40 methanol/water for this separation. The choice of the solvent can affect the resolution of the AHL's, so it would be interesting to compare several references to see what people are using. Do you have any methanol available, so you can compare ethyl acetate to methanol/water? Do you have an enclosed chamber of some sort to hold the microscope slides to develop the TLC? You will also need some sort of AHL standard to use. These may not be commercially available, so one possibility would be to contact one or more of the authors of the recently published papers and ask for a small amount of standard. You can also ask the authors about details of their experiment; since they have done the experiment, they are the best source of accurate information. You would acknowledge the gift in your acknowledgement section of your board. Let me know if you don’t have this particular reference; I can upload it for you.

On the TLC slide, the different AHL’s will migrate according to their size. The molecules with the smaller carbon chains will migrate faster than the molecules with longer carbon chains. You need at least one or two of the standards so you can identify the spots on the TLC because there will probably be extraneous non-AHL’s appearing in your sample.

4. I did notice that the experiments are done in broth culture, and I think it would be easier to measure the concentration of AHL’s in a liquid medium. For a liquid medium, you would just make up your LB medium without agar and call it LB broth. However, you would need a glass test tube with lid to hold the broth culture. Do you have anything suitable available? I noticed that one reference used chloroform to extract AHL’s from the broth medium. Do you have any chloroform or other non-polar solvent to extract the AHL’s.

5. I agree with Terik, that you do have time to get results after the winter break. Your initial results will be very helpful in deciding what you will do for the next experiment. And you are correct to concentrate on gathering all of the materials you will need for your experiment this week.

I think I owe you a few answers, so I will reread your questions and post another reply tomorrow.

Donna Hardy

[Trader]

Thank you SO MUCH!

All the concepts from the research were previously muddled, but they are a lot clearer now .

I'll update very soon with my findings

1. Researching...and I found a peer reviewed article that explains almost everything, including word for word "Construction of a GFP-based AHL sensor" I'll attempt to decipher what it's trying to say.

2. It'd be so interesting to find out which one's more effective! Thanks =)-- you've explained it in a much more concise way than the fancy articles

3. Wow -- interesting. I also found a research article that also uses methanol/water with a 60:40 (v/v) [I want to say v/v = in vivo...?] concentration ratio. We have the methanol/ethyl acetate, and it looks like it's commonly used so it's good =)

Hopefully I'll find more references so that I'll know which one is more effective. As for the enclosed chamber (sorry) but I am still attempting to understand the process of using TLC that I'm not sure where the enclosed chamber would come in. My teacher did say that we would have something that can hold the slides while it is being prepared. Also in the same research article it mentions that

The presence of AHLs in the extracts was tested by C18 reverse-phase TLC (Uniplate RPS Reverse-Phase Hydrocarbon Impregnated Silica Gel) -- so is it true that would making the TLC plates still be required if we could somehow access these "prepared" TLC plates?

As for the AHL standard, I have a few references to start with -- thanks for the idea

4. I've seen some of the glass test tubes. Would this be anything related to the "Erlenmeyer flask" -- I've also seen open glass test tubes in our lab -- I'll ask if there are closed ones. It looks like that if I'm to use a liquid culture, I'll have to have a shaker and analyze the supernatant right? -- or am I confusing myself O=, though I have seen the term "shaker" and "analyzing the supernatant for AHLs" many times throughout the experiments.

5. Thank you for the confidence boost

I'm going to update this materials list as I go:

• For LB media/broth (1 L)
  - 10 g tryptone
  - 5 g yeast extract
  - 10 g NaCl
  - 15 g agar
  - Distilled water
  - Measuring cylinder (200 ml +)
  - Autoclave
  - 2 L flask (that can be tightly shut, designed for autoclave)
  - Petri dish X 10 (? I know that bacterial strains can easily get infected, but would 10 be enough? (I’m hoping to get more trials for more accurate results)
  - Scale boats (weighing) + Scale
  - Oven mitts/tongs
  - pH strip/meter (detecting appropriate pH for optimal bacterial growth)
  - Sodium hydroxide/TRIS for ‘boosting’ Ph
  - Bacteria strain [teacher is checking if I have access to v. fischeri -- if I don't, my backup is e. coli]
  - Cotton swabs for inoculation
  - Shaker/centrifugal IF liquid broth
  
  Materials for the TLC
  - Microscopic slides (for the plates)
  - Silica (solid absorbent)
  - Erlenmeyer flask (for preparation of silica) - 500 ml
  - Ethyl acetate and methanol
  - An enclosed chamber of some sort
  - Glass tubes that can be tightly sealed.
  - A-HL standard
  

  I've seen in here that a "Uniplate RPS Reverse-Phase HydroCarbon Impregnated Silica Gel) is used > In your experience, would experiments like these require "specially made" silica gel, or can we "make" them ourselves? Thanks!

  Materials for the bioluminesence bioassay
  - A ... Camera (?) for capturing the bioluminesence...
  - Not sure yet, more research needed.
  
  Materials for the sensor strain
  - A strain of bacteria classified as a "sensor strain" > More research needed
  
  Bold items are ones that I need more research on, and/or checking if I have access to or attempting to find a way to get it.
  
  Update: I've removed the boldings for the materials I do have. These I "do have" means that we have them in our lab and they are ready to use

I know my experiment will involve TLC assay, bioluminesence assay, a sensor strain bioassay, comparing the techniques. THIS IS SO EXCITING!

Though... my teacher suggests the following:

After reading the method for the bioassay for AHL, I'm certain we wouldn't have everything in time to have reasonable results by March.
I'd suggest the following phases.
Phase I: This year, do a study with bacteria (an environmental factor like CO2, anti bacterial study, or "Mythbusters" type study such as testing the statement, "bacteria can go through 10 layers of toilet paper") This would give you experience in bacteriology techniques.
Phase II: Next year, analyze for some kind of extract from bacteria using TLC. Perhaps extracting the bioluminescent molecule from bacteria. This would continue your bacteriology techniques plus learning extraction and separation techniques.
Phase III: Senior year, do a bioassay such as for AHL.
How does this plan sound?

----
Hmm...would it be possible to skip phase I and get onto phase II? Based on your experience, what do you think I should do? o=...I didn't have the impression that doing a bioassay would take too long, as one experiment I have here completed a bioassay in 7 days...

And one more kind of unrelated question: Is it normal to have high school students have experts help them step by step? (I hope I'm not the only one! =))

Thank you for your patience in answering my questions (some which appear to be a bit obvious but I really can't find the answers in the articles =P)!!

[deleted-2131]

Hi Trader,

The person who is familiar with TLC should be starting to join the dialogue here in the next few days, so hopefully she will be able to provide another perspective to help understand that aspect of the project. With regards to your question about having a mentor/expert to help you, the truth is that most of the students who do a high-level project, such as yours, do have a mentor of some sort, whether it be a university professor who is over the lab they are working in, a subject matter expert they visit with occasionally, or an Expert such as Donna Hardy who serve on these Forums. A mentor isn't there to do the work for you, but s/he is there to answer questions and help you resolve problems as they arrive. They are a guide on the side while you do the majority of the work. A good mentor-student relationship goes a long way into helping a student be successful at the top levels of competition. You might find this article helps you understand the role of a mentor: https://www.sciencebuddies.org/science- ... tors.shtml.

It sounds like you're making all kinds of progress right now, so keep the questions coming and keep working hard!

[donnahardy2]

Hi Trader,

I read another article last night, so I am slowly coming up to speed on this topic. Thanks for posting your references; I'll look through these tonight.

Here are some additional considerations about your proposed experiments, mostly information on the chemistry.

1. Ethyl acetate can also be used in the place of chloroform to extract the AHL's from a broth culture of the bacteria, and would be safer to work with, although you would still need to follow safety precautions for working with solvents. The technique for extracting the AHL’s is called solvent extraction or liquid/liquid extraction and is based on the difference in solubility of molecules for an aqueous and organic solvent. Here’s a website that explains the basic principle, http://en.wikipedia.org/wiki/Solvent_extraction. And, yes, you will not use this as a reference for your science project, but the information on this posting is excellent.

2. Extracting AHL’s. If you add a non-polar solvent such as chloroform or ethyl acetate to a liquid culture of bacteria producing AHL’s, and mix it very well, all molecules that are more soluble in the aqueous medium, such as proteins, nucleic acid, carbohydrates, and amino acids will stay in the aqueous phase. Hydrophobic molecules that contain lots of carbons and hydrogen like the AHL’s and the cell membrane will dissolve, or partition into the non-polar solvent. This step will help concentrate the ALH’s will give you a partially purified sample for the TLC plates. Go to one of your articles that include a structure of an AHL. You will see the homoserine amino acid linked by an amide bond to carbon chain of 4 or more carbons. Some of the AHL’s have an oxygen atom on the carbon chain and these AHL’s have an “oxo” in the name. The carbon chain of the AHL makes the molecule hydrophobic, so more soluble in organic solvent. Understanding the chemistry of this project is going to be very important, so please let me know if you have any questions about this.

3. Agar cultures. You will definitely be doing agar cultures once you get to this stage of your project. The biosensor strains of bacteria do not have the ability to produce AHL’s, so these are detected by growing the biosensor strain on an agar plate close to the test strain. If AHL’s are produced by the test strain, they will diffuse through the agar and the biosensor strain will then be able to produce the pigment or other function that requires the presence of AHL’s.

4. TLC. The development of the TLC plates for these experiments is very interesting; I was completely unfamiliar with the technique until I read about it, but it does make sense. The AHL’s are separated on the TLC plate with the methanol:water mixture and then the plate is overlayed with agar containing a high concentration of the biosensor organisms. The AHL’s are detected when the biosensor organisms grow and produce the pigment in the agar overlay.

5. TLC silica. The traditional TLC silica uses fine particles of silicic acid (H2SiO2). The silica used in the papers I have seen so far has been silicic acid that is bonded to long chains of 18 carbon with hydrogen. The bare silicic acid gives a polar surface for the sample to interact with; the C18 silica would have an almost completely hydrophobic surface. Since the AHL’s have carbon chains that vary in the length of the carbon, they will have a varying degree of hydrophobicity, and it makes sense to use a C18 silica for the application. If, for example, you had an AHL sample containing a C4 and a C10 AHL, the C10 AHL would have a stronger attraction for the surface of the C18 silica so would migrate more slowly compared to the C4 AHL. If you can get the prepared C18 plates, you would not have to prepare your own plates, and your results would be more consistent and reproducible. I’m glad we have a TLC expert who will be joining us in the near future, as this will be very helpful to explain more details of this technique.

6. 3-year plan. Your teacher is very experienced and wise. I agree with her completely. Doing a preliminary project this year just to learn the basic techniques will be a challenge and will help you become an expert for a follow up project. You will be amazed at what you can learn when you actually start to get results in the laboratory. Every experiment will give you ideas for 10 more experiments, so you will have to focus on your goal to stay on track. “Can bacteria go through 10 layers of toilet paper?” is an excellent project to start with and you will learn all of the techniques. However, you are going to need a biosensor strain to detect the AHL’s. Have you written to any of the authors to ask for a sample yet? While you are doing, this, you might ask any authors in your geographic vicinity if you can have a summer job. I had an intern position at a local University lab between 10 and 11ths grades through a local science program, and it was a great experience, and your project would benefit from an opportunity like this. But, you will need to ask about this possibility.

This is a great project. Let me know when you have more questions.

Donna Hardy

[Trader]

Wow! Thank you Donna Hardy for answering these concept questions!

I wrote the letter to two authors of a relevant research article and found a source:

The thin layer chromatography is a technique for the separation of different molecules depending on the retention factor of the molecules (Rf) with the use of a solvent phase going through a silica plate (in my case methanol diluted in water to the 60%). You can buy different AHL standards to different companies (like SIGMA-Aldrich), but they used to have only non-hydroxilated molecules.[1] If you want hydroxilated molecules there is a group in England leaded by Paul Williams ([email protected]) that are synthesizing them and selling them....

[1] My research tells me that hydroxylated silica would be oxidized (and therefore would have "lost" electrons/hydrogen and gained oxygen) -- I seem to not be able to find any articles giving a reference

To TLC expert: From your experience, how would the oxidation state of the silica affect the solvent/solute "migration" and its results?

>> Thanks!

A similar question is, as I've seen TLC being used to detect AHLs many times, there is a high chance I'll be using b. subtillis, which uses ComX QS signals (not AHL) -- I'm afraid I can't find any previous experiments done using TLC on b. subtillis. So,

Would be correct to say that TLC is able to separate AHLs due to its common structure as a signal and therefore TLC could also separate other quorum sensing signals including ComX QS signals in the b. subtillis that I will be using?

4. Cool! Then...

Just to clarify: the use of TLC can be used with both test strains/sensor strains cultured in agar plates as well as a liquid broth right?

One last thing -- I think I understand the process of using a sensor strain a lot better now, though...

Since after the test strain reaches certain population density, the AHLs are "transferred" to the sensor strains, I'm still not quite sure of the role of using the TLC in this process -- since we can see from the effects of bringing the sensor strain in contact with the AHLs (such as bioluminesence in case of v. fischeri), why would we need TLC to separate the signals in between?

6. I agree with my teacher though [see post below]

Thank you all the experts, Donna Hardy, Terik Daly and everyone else who beared with the long lists of questions I've had previously. Also thanks for helping me with the TLC expert as it would be a great help to the experiment. I'll be sure to read through the research regarding the TLC, apply it to the experiment and hopefully get started after winter break! (Jan 5)

[Trader]

Update: Sensor strain "question" solved >
There is a way! As bacteria go through 3 phases of growth, lag phase, log phase and stationary phase. For v. harveyi for example, 5 - 5.5 h after inoculation, there is minimal creation of AHL. Experiments either use this as the "sensor strain", or can use this in place of a sensor strain.

Now I can focus on what to test, as I'm pretty sure about what to do with the procedure (though there are still some "why?" questions I'm not sure about [above]). My question to myself now is "what affects the communications between bacteria?"

[donnahardy2]

Hi Trader,

It's good that you are thinking about the details of your experiment. You have a valid concern about the detection of the AHL's. I will think about this and post a more complete reply tomorrow morning.

Donna Hardy

[deleted-71490]

Trader:

If you have a university close by, I suggest a visit to the library and look at current textbooks on bacteriology/microbiology. Also visit the biology/microbiology department, describe your ideas and ask all your questions about bacterial communication.

You are doing a good job of narrowing the topic.

Good luck,

Matt Mulanax

[donnahardy2]

Hi Trader,

It’s hard to keep up with you, because you are progressing so quickly on your project. I’m sorry I haven’t had time to respond to all of your questions for the past few days. Here, finally, are some responses to your last two postings:

1. Standards. Here is the information on AHL standards from the Sigma website. http://www.sigmaaldrich.com/analytical- ... crobiology. I like the information from Sigma because it includes the structure of the molecule. Take the time to look through and notice the similarities and differences in the molecular structure of the different AHL’s. What is the relationship to the name and the number of carbons in each molecule? Please notice how expensive these standards are, for example N-butyryl-DL-homoseine lactone is $57 plus shipping for 25 mg. If you can purchase just one of the standards, it will work as a control for both agar plates and the TLC chromatography. You just need to pick one that your sensor strain is sensitive to.

2. Hydroxylation. I think the reference to hydroxylated molecules refers to the AHL’s, not to the silicic acid.

3. ComX QS in B. subtilis. Bacillus subtilis is a Gram-positive bacterium, completely unrelated to the Gram-negative organisms that use AHL’s for quorum sensing. This organism uses a 6-amino acid peptide for QS. Peptides are usually analyzed by reversed phase HPLC techniques, so you would not be able to use the AHL’s to detect QS in this organisms. .

3. Chromatography theory. I am looking for a good reference on the theory of TLC for you, but I haven’t found one yet. You can find some information by searching for “chromatography of lipids,” “silicic acid chromatography,” and “reversed phase chromatography.” It won’t matter if the information is for thin layer chromatography or column chromatography, because the theory of separation is the same. You can also refer to high school chemistry textbook and look for information on lipids, polar, and non-polar.

The AHL’s are polar lipids. The oxygens, hydroxyl groups (OH), and nitrogens give the molecules a polar character. These molecules can interact with silicic acid (H2SiO3 is an example of one silicic acid) by polar interaction. The silicic acid or silica gel, is the traditional solid support that is used for TLC plates for separation of polar lipids.

All lipids are hydrophobic because their chemical structure includes carbon and hydrogen chains. The number of carbons and hydrogens in each molecule can be used to separate the individual molecules based on their relative hydrophobicity. An AHL’s that contains 16 carbons will be more hydrophobic than one with 4 carbons. Since the AHL’s have similar polar structure, but vary in the number of carbons, they are usually separated by interaction with C18 reversed phase silica. C18 is the number of carbon/hydrogens that give beef fat it’s character, which means that C18 silica (18 carbon atoms) is really hydrophobic. To give you a point of reference, butter contains a softer C4 lipid so is less hydrophobic. All AHL’s will bind to C18 silica and the very polar solvent (methanol and water) will elute the less hydrophobic AHL’s faster than the more hydrophobic AHL’s. Here is a question. If you were doing TLC of AHL’s, starting from the bottom of the C18 silica plate and eluting upwards, would a C4 AHL migrate faster or slower than a C16 AHL?

4. Agar plates vs. broth cultures. Agar plates are used to detect the presence of AHL’s; broth cultures are used to identify the specific AHL.

AHL’s are usually analyzed from broth cultures because a larger volume of culture is apparently necessary to purify enough of the AHL’s for the TLC analysis. TLC is used to identify the specific AHL present because there will a separation of the AHL’s on the C18 silica and the distance of migration (Rf value) can be compared to a standard of known identity.

Agar plates are used to detect the presence of AHL’s in a test strain by growing it close to a sensor strain. The AHL’s are fairly small molecules that are released extracellularly so they will diffuse through the medium to the sensor strain, which will produce its pigment only when AHL’s are present.

Your use of the agar or broth cultures with TLC analysis will depend on the experiment that you design. What do you want to accomplish? Which experiment are you going to do?

Let me know if you have any more questions.

Donna Hardy

[Trader]

Oh my… I’ve finally understood the TLC concept!! So basically it is a “race” between the different AHLs right, with the most hydrophobic (with the most carbons) migrating the farthest, while the least hydrophobic (but still hydrophobic) AHLs migrating the least! This is so cool!

And I’ve FINALLY understood the difference between the procedures required for AHL detection and AHL identification –

THANK YOU SO MUCH! I feel like I have a direction now!

Oh my…I don’t know if a $57 is within my budget )=. Is it possible to … “manufacture” AHL standards?

I really want to use TLC after finally understanding it, in addition to all the time and effort you have spent teaching me, a slow high school student =P, how the polar interactions work between the lipids and the silica! )=.

I could end up writing two research plans, and ending up choosing one of them on the day that I discover whether or not I have access to a) v. fischeri and b) AHL standards. That's it. If it's not within my budget I'll make it within my budget >=(.

Possibilities:

NO AHL standard, NO v. fischeri – e. coli v. b. subtilis, comparison b/w AMOUNT of AHLs that pass through a given factor, i.e. amount of glucose. Could be interesting as there is gram negative e. coli and gram positive b. subtilis for sure. Comparing the two might be hard though, because it would be e. coli sensor strains’s “cell division” (as that is what the e. coli uses QS for), and the “sporulation” in b. subtilis. I’ll have to set up a lot of controls to actually test the intensity of the amount of signals (since I won’t have the AHL standard to use TLC)

YES AHL standard, NO v. fischeri – YAY! I can use TLC! I can test the different types of signals that pass through a given factor, i.e. amount of glucose inhibits which signals and boosts which other signals between the two different strains, building on the sensor strain method and ultimately getting not only the amount but also the type of AHL.

NO AHL standard, YES v. fischeri – expand idea #1 to cover 3 different types of systems? V. fischeri is very useful however, for its bioluminescence.

YES AHL standard, YES v. fischeri, BEST CASE SCENARIO -- a combination of idea 1 and 2.

I think I'm ready to write out the process now! Of course, there are some mini concept questions, but I think I can Internet my way through. Though, I do still need to think of a factor "X" that affects either the QS or the detection of AHLs.

Thank you so much! I'll update this when I have a seemingly unsolvable problem in setting up the experiment

[donnahardy2]

Hi Trader,

Here is some additional information and suggestions:

1. TLC concept: You almost have this down, and your racing analogy is excellent. The short chain AHL’s have a lower selectivity for the C18 silica compared to the higher chain AHL’s so will migrate faster on the TLC plate. The higher chain AHL’s are “stickier,” so they move more slowly. So the short-chain AHL’s will always win the race on the TLC plate.

2. It’s going to be difficult to do this project without at least one AHL standard and one sensor strain. Have you had a response to any of the researchers that you wrote to? If so, then write again and ask how you can get a sample of a sensor strain and a sample of an AHL control. It doesn’t have to be V. fischeri, any sensor strain will do. If you haven’t had a response, then write again, and write as many authors as you can locate. Generally the research papers will have the e-mail address of the primary author so that is the best person to contact. Many academic laboratories are officially closed for the holidays, but many researchers will work through the winter break because it’s a good time to get lab work done. It never hurts to ask for something that you need, and many researchers are happy to help high school students.

3. You can call Sigma technical support department at 1-800-325-5832 (US number) and ask if it would be possible to get a sample of one of the AHL’s. Make sure you have their website up so you can refer to the catalog numbers you are interested in. If you don’t get a positive response, ask if they have an in-house expert on this subject and see if you can talk to that individual and at least get more basic background information on your project. Since Sigma has recently started offering AHL’s, they may have someone with expertise on this subject, or some literature available. I’ve never asked for a sample from Sigma, but some companies are willing to send samples, so again, it won’t hurt to ask.

4. Scientific supplies are expensive, and most researchers have grant money available to pay for supplies. Have you ever asked your teacher if there is any source of funding for science fair projects? If there is no extra money available this year, you may need to find a suitable grant to apply for next year.

5. Synthesizing or purifying an AHL and developing your own mutant sensor strain would be complete projects on their own. Why don’t you explore your other options to obtain the necessary supplies before considering this?

Donna Hardy

[deleted-2131]

Hi Trader,

Donna is right; you are going to need to find some way to pay for supplies. I know that there are several organizations that provide funds to students/schools who have science fair programs. In addition to talking with your teacher, I would also suggest contacting someone who is involved in the administrative side of your fair (often called the fair manager); they may be able to point you towards such an organization that may (or may not) exist in your area.

[Trader]

Merry Christmas! (It is here anyways )

I knew that -- now that I get it, I'm going to try to find that AHL standard even harder ! I am not sure though about the policies with contacting scientists in science fair projects as this is my first time -- does asking them for an AHL standard mean getting it for free after thez 'send' it over? (Sorry my question is a bit awkwardly phrased)

I've asked several authors and some replied with "You can buy them from Sigma", so I'm not sure if by 'asking' I should reply and ask "Thank you for the source -- I've checked out the website and found that the expenses are somewhat beyond my budget and I am most likely unable to have access to a science fair project grant this year. Would it be possible for you to give me an AHL sample that you used in the experiment you mentioned in this article?" -- Sorry, it seems like a very obvious question with an obvious answer, yet this is my first time doing anything like this =P... thanks for understanding.

As for the sensor strain,

Wouldn't the access be relatively easy, as we can have the indicator strain inoculated first, and then have the sensor strain inoculated later such that the stationary phase (where the sensor strain would have minimal QS signal production and then treat it as the strain that 'cannot produce its QS signals's?

Though I would have to have my controls carefully set up, because there is going to be a tiny variation there.

I will continue to write to all the authors who have written an article in the past few years saying whether I can have a free sample of the AHL standards they have used...

I've seen in some peer reviewed articles of pictures of their TLC experiments... I can look into those, but if the 'discussions' base their results on those, it seems like their result (the picture) would be close to the actual standard. Would it be okay to use those? I asked many of the authors with those pictures for a sample, but I guess the winter break is not having many respond. But I must get that AHL xD.

What is somewhat awkward is that I don't know whether our school supply stores would have access to AHL standards. There was a recent experiment using e. coli and bioluminesence and quorum sensing, and hopefully with that they used any AHL standard related to e. coli that I can use. I won't have access to my science teacher until after break -- should have asked for a contact number ):

In the meanwhile, I'll contact Sigma and the organizations. Would it be possible for you to give me some of the names to those organizations?

I've managed to narrow down my topic to a research question that is something around "How does the chemicals found in 'common medium' used to culture bacteria in experiments affect the process and/or detection of quorum sensing signals in e. coli and b. subtilis?"

I've read about how bacteria themselves release ... oxidized halogens which interfere with AHLs, and thought that perhaps there would be similar chemicals found in the media that microbiologists use all the time that affects with the chemical signalling. It appears that glucose and galactose for example, each has their special effect on the detection of AHL signals via the sensor strain. I've tried to gather as many articles regarding the inhibition of detection of AHL signals and so far I haven't found an article approaching this topic, though I was wondering if

Is it known by the microbiology society in general exactly what everything in the medium they use and how it affects QS signal detection that that's why there are no articles on it, or that it is common knowledge (except to me ):) that the chemicals used in medium chemically will not interact with signals?

Time to email

THANK YOU so much!

[donnahardy2]

Hi Trader,

Thanks, and Merry Christmas to you as well!

Your idea to inoculate the agar plates at different times to detect the AHL production is a clever idea. You could certainly try this, but I would be concerned that the sensor strain would not be visible early enough to detect the difference. You haven’t grown bacteria yet, but after inoculation, they can’t be seen during the lag and log phases, and appear as visible colonies on agar plates or turbidity in broth only after incubation for 18-24 hours. The appearance of the AHL-dependent function usually appears at the end of the log phase of growth, so you might miss on the timing. So, your experiment might work, but you might not be able to see the results.

You should check out the advanced chromatography project on the science buddies website. It gives a good explanation of the general principles of chromatography and how to calculate Rf values. The specific experiment is on paper chromatography, but the general principles would apply to your project: https://www.sciencebuddies.org/science- ... k&from=TSW

AHL standards and C18 reversed phased TLC silica are not general lab supplies, so I’m certain you will need to order these items. Here are a couple of websites that offer this type of product. Looking at the price for the Merck plates, I think that you will need to contact these companies and ask for samples if you can’t find a source of funding for this project.

http://www.vgdusa.com/EM_Merck_TLC_Plates.htm
Silica Gel RP-18 F254 TLC 5 x 10 250 Yes — EM-15685-1 Pack of 25 $305.99

Here is another company that offers this type of product:
http://www.whatman.com/PartisilReversed ... lates.aspx

Sigma is the only company I can find that offers AHL standards. Please note that you really need to identify your sensor strain before you can request a specific AHL standard. You can also contact ATCC, the American Type Culture Collection, http://www.atcc.org/CulturesandProducts ... fault.aspx, and ask if they have any bacteria that can be used as AHL sensors. You should also contact the American Society for Microbiology, http://www.asm.org/. I noticed that their home page has a “find a mentor” selection and they have 400 microbiologists available to help. Also, ask how you can contact your local branch of the American Society of Microbiology, as that will put you in contact with microbiologists who are close to you. If possible, do plan to attend a local ASM meeting to meet local microbiologists in person. Make a list of what you need (AHL sensor bacterium, AHL standard, C18 reversed phase TLC silica or plates, grant money, and a local mentor), and ask each contact for the appropriate help.

Your research question is interesting, however, I think that you may want to think more about what you really want to do. Bacterial media consist of inorganic salts, peptides and proteins, and glucose or another carbon source. Specialized bacterial media may have other ingredients to support the growth of specialized microorganisms. For example V. fischeri is grown in a medium that contains the same concentration of salt as ocean water. Bacteria can grow in the medium and will produce the AHL’s, which are composed of carbon, hydrogen, nitrogen, and oxygen, at the right time without any special care. I suppose that conditions that provide the optimum conditions for growth would provide the optimum conditions for AHL production. What were you thinking about doing for a specific experiment on this project?

I thought your idea of “can bacteria communicate through 10 layers of toilet paper?” was a good idea. I have not seen any references that answer the question of the distance that bacteria can communicate with AHL’s. It would be interesting to find how far the AHL’s can diffuse through medium to communicate with the sensor strain. This would be a unique project.

And your idea about the competition between bacterial species is a great topic. This reference is a study on the competition between the Gram-positive Bacillus thuringiensis and the plant pathogen Erwinia carotovora. http://aem.asm.org/cgi/content/abstract/70/2/954 Interestingly, the Bacillus secretes an enzyme that interferes with AHL production, which affects the plant pathogenicity, but not the growth of the Erwinia. Co-culturing an AHL producer and a Bacillus species could be the basis of a very interesting project.

Donna Hardy

[Trader]

Cool, I never thought about how we wouldn't be able to see the bacteria until later...I'll need to see the results in order to detect whether there are AHLs there or not -- I thought I saw e. coli used as a sensor strain somewhere -- even though we wouldn't be able to see it, would using a microscope work, for example? (trying to find ways to 'substitute' for a sensor strain ).

The TLC silica is one thing that I have already confirmed that I can have and for the AHL standards I have asked for 'samples' (this does mean asking them for a 'free sample' right? Sorry-- I haven't really done this kind of a thing before)

I think the toilet paper project is cool too -- originally I had put it on 'hold' as I wasn't sure how I could put the 'toilet paper' part of the experiment into the experiment, but the idea of distance is very interesting. ... Now that I think about it...yea! . Yea, I've thought a bit about the medium idea and it seems like those are some concerns that might make it not so good of a project, though what I really was trying to do there was analyze the very cool 'quorum sensing system interference' that I read about and you mentioned (thanks for the reference!)

I shall continue to email and poke around

[donnahardy2]

Hi Trader,

Yes, E. coli could work as a sensor, but it would need to be missing the AHL synthetase gene and have a plasmid that would express a characteristic such as green fluorescent protein, that would respond to the presence of AHL.

Are you sure you have access to reversed phase C18 silica for TLC? The traditional silica for TLC is not bonded with C18 groups, and the C18 silica used for separation of the AHL's is much more expensive than silica gel. You should double check on this. For the sample, yes you will be asking for a free sample. It is very common for researchers to ask for samples of expensive reagents to find out if the product will work for the research application. The implication is that the researcher will buy a lot of the product if it works. You are asking for a free sample because you are doing a high school project and you need the sample to do your experiments. However, you are also planning to do a prize-winning project, and you will acknowledge the gift of the sample in your presentation and give the company providing the sample free publicity. Asking for free samples is best done by calling the company on the phone and trying to find a sympathetic company representative that can help you.

You are right about the medium idea; it would really make a good project. But a science fair project with a practical application will be more appealing to the judges. You could also work on practical applications such as milk or food spoilage, acid mine leaching (biofilm formation, or a plant disease.

Donna Hardy

[Trader]

With regards to the e. coli ... Apparently the biology class in my school recently did an experiment with a genetically modified e. coli with a bioluminesence gene. I can use that instead for a sensor strain (I suddenly thought about this), though I'm not sure if it would have that AHL synthase gene.

I thought e. coli normally produced a certain type of AHL that, if the sensor strain is e. coli with bioluminesence, that it would work as a sensor strain and wouldn't need an additional AHL synthase gene added in?

However, for detection of QS in signals in b. subtilis, I might still need to use the timing method and test for sporulation, though I have an article that I just downloaded. Hopefullz I can find some techniques and more background information about b. subtilis.

I could double check for the reversed phase C18 silica...in Jan 4, but I remember that when I told my teacher about wanting to do a project on TLC and showing the possible materials that I might use and there were apparently no problems. I see what you mean though...if am allowed to order the silica, chances are I can also get a sensor strain and an AHL standard, though that would take another 2 weeks perhaps. That time, I didn't know that C18 silica is one of the expensive items, and it wasn't until now actually, that I realized that =P

By milk or food spoilage, do you mean how chemicals in that inhibit QS signalling in bacteria? That could be interesting.

I'll make a list of all the ideas floating around, though the toilet paper idea is somewhere near the top, and it applies to the 'QS signalling inhibition' topic I want to pursue.

Thank you

[donnahardy2]

Hi Trader,

The E. coli used by your biology class has the bioluminescence plasmid, but it also has AHL synthase, otherwise you would never see the green pigment forming. If you had this E. coli without the AHL synthase gene, it would work as a sensor strain because it would only produce the green fluorescent protein if exposed to AHL’s from an outside source, such as a test organism.

I have been thinking about your TLC application, and I’m sure you would be able to use the regular silica gel to detect the AHL’s (if you had a sensor organism). The AHL molecules have a polar end, so will separate on regular TLC silica. The reversed phase silica is necessary to separate the AHL’s with carbon chains of different lengths (C4, C6, C8, etc. Various AHL’s will probably co-migrate because they have similar polarity, but you would certainly be able to obtain qualitative results with regular silica and definitely detect the presence or absence of ALH’s. So, don't worry about getting the C18 silica, but be sure and design an experiment that won't require identification of specific AHL's.

The information on the Gram-positive Bacillus subtilis does not seem to directly apply to an investigation of AHL production by Gram-negative organisms like E. coli. This reference uses several cloned strains of B. subtilis and requires extensive knowledge of cloning and biochemical pathways. Why don’t you concentrate on doing a basic project like distance that AHL’s? Or, maybe find out when AHL’s appear in milk that is spoiling?

Donna Hardy

[Trader]

Yes I checked out the reference and it turned out that it wasn't too useful.

I just want to clarify something:

So sensor strains differ only from a "normal" strain in terms of its inability to produce the signals, meaning its lack of a QS signal synthase right?

Cool! Thanks for the information regarding the regular silica. This would help a lot if I can't get the C18 silica. Since using regular silica would mean that I wouldn't have to identify the AHLs, I would still need an AHL standard though right? (So far negative responses, perhaps its the winterbreak...)

The distance and the milk both are very interesting. I haven't had access to internet in the past few...36 hours-ish, and I'm afraid I haven't done much research, but if I'm not mistaken, e. coli would be involved in the milk spoiling right?

I'll update tomorrow =)

[donnahardy2]

Hi Trader,

Yes, the sensor strains have everything that the normal strains do, except they don't have AHL synthase, so can't produce AHL's. Without AHL's there is no quorum sensing signal to produce a pigment or other characteristic (plant disease, spoiled milk, biofilm) that is dependent on AHL production.

Milk-spoilage organisms have names like Pseudomonas, Alkaligenes, and Flavobacterium. These organisms grow well at refrigerator temperatures and are known as "psychrotrophs," (cold-loving). Wild-type E. coli is a member of the normal flora of mammalian intestinal tracks, so it grows optimally at 37 degrees Centigrade. All Gram-negative bacteria produce ALH's, so if you did your experiment on milk that was spoiled by Gram-negative bacteria, you would expect to detect AHL's. Bacillus species can also cause milk spoilage, but do not produce AHL's.

Donna Hardy

[Trader]

Interesting. I could investigate the different processes involved in milk spoilage b/w e. coli and b. subtilis, especially with e. coli since it involves AHL...if I take this direction, I would not have to use an AHL standard right?

Perhaps I could first do this experiment, and somehow make some time so that I can gather my other materials (AHL standard, C18 silica !).

For the sensor strain, if I use a microscope (for e. coli cell division) or possibly, use the "timing method" for the e. coli with the bioluminesence gene, it could work to the point that I could avoid having to get a sensor strain altogether?

This way I can focus on AHL standard and C18 silica. If I could just reach my teacher and see if I have access to those supplies (because our biology class has been doing TLC related experiments lately...they should have them <_<)...

I'm still going to hold onto the idea of AHL v. distance and toilet paper, because IF (it's a big if, but...) I can have access to the AHL standards, the C18 silica, then I can do it!

It would be cool to find out what types of signals manage to go through which distance due to its structure. Plus, after understanding how TLC works, I'm going to try to fit this in.

The media idea is probably not practical, but

If the research question was: "The effect of (some chemical in the media) in the preparation of cell-free culture fluids", would that be practical?

What I'm really trying to do is play with the idea of inhibiting AHL signals. I know I'm switching around ideas a lot, and I guess I'm searching for the "right" one with one that deals with quorum sensing. =)

Thank you!

[Trader]

HAPPY NEW YEAR everyone!

[donnahardy2]

Hi Trader,

Happy New Year to you too!

I assume you have not had a reply to any of your inquiries yet. Hopefully, someone will return from the holiday break and send a positive reply to one of your requests. You could do a project if you could just get a sensor strain to work with. Otherwise, your project for this year might have to be exposing a bacterium to a mutagen and isolating your own sensor strain. This would be a lot of work, but doable.

Donna Hardy

[deleted-2131]

Hi Trader,

Hapy New Year to you, too! Keep us posted on your project and keep up the tenacity; hard work will pay off!

[Trader]

Yes, I'm afraid the sensor strain/C18 silica/AHL standard search isn't making much progress.

Would exposing the bacteria to a mutagen eventually make it lose its ability to create AHLs and therefore create a sensor strain? (Though just to confirm: would it be just as "doable" to use the "timing" sensor strain method?)

Right now I am going to continue with the AHL and distance idea, using normal silica and the timing sensor strain (possible ways to make it workable -- microscopes?) to see the "maximum distance" which sensor strains can sense the AHLs.

This way, I'll at least have something to start with =D, and in the meanwhile, HOPEFULLY I can gather my AHL standard, C18 silica and a better sensor strain through asking for samples or from my school's bio supply.

FINALLY time to start! xD!

Update: Seems like the AHL v. distance isn't really applicable and practical (which means I'm going to change the RQ again ), so I've decided to involve the use of AI-2 (supplied by e. coli) in the b. subtilis processes.

It's very interesting because b. subtilis seems to be a model organism for metabolism of carbon sources (which may lead to a question of how careful preparation and adaptation to the bacteria's growth is required in agar plates and/or liquid cultures) and secretes industrially important enzymes. Cooool.

Can an RQ be something like "What is the effect/role of AI-2 in b. subtilis metabolism of carbon sources/secretion of industrially important enzymes"?

The project title can be something like "Inhibition of AI-2 in b. subtilis leading to..."

xD!! The RQ expanded from the milk spoilage idea -- I was thinking that to combine QS and something practical might be something like this.

[donnahardy2]

Hi Trader,

All of the inquiries you have sent have gone to professors at academic institutions and all of these people tend to be out of town during the holidays. Please be patient for a few more days and hopefully you will receive a positive reply from someone. You might want to politely resend your e-mail messages on Sunday/Monday so your inquiry will be at the top of the list again. It’s hard to wade through two weeks of messages when you’ve been out of town, and your messages from before Christmas will be at the bottom of the list.

You always have lots of good questions, and I admire your perseverance on this project, as well as your flexibility in waiting to make a decision on your final RQ. Here are some comments:

If you expose a bacterium like C. violaceum that produces a pigment to a mutagen like UV light, there will be lots of random mutations. If you grow the mutagen-exposed culture on agar plates, and then select for non-pigmented colonies, you might have an AHL-negative mutant. You would need to do additional testing to determine why the non-pigmented colonies have lost their ability to produce the pigment, but, if successful, you would have your own sensor strain. This project will require lots of Petri dishes, agar, time and patience, and there is no guarantee of success, but it is definitely a possibility. If you decide to go this route, I will consult with my molecular biology colleagues and get more information to send to you.

Why isn’t the AHL v distance applicable and practical? You would just need a sensor strain and test strain to do this.

If you switch to a project involving the effect of AI-2 supplied by E. coli on B. subtilis, you will still need to have the microorganisms. What industrial important enzymes were you going to study? You would need a way to measure the bacterial activity you would be studying. Bacillus species are a source of the antibiotic, polymyxin, and an enzyme, alpha-amylase. A practical project like the milk spoilage project would be appealing, but I’m not certain how you would do this. Have you thought about the experimental materials and detailed methods? You probably need to have two or three options to consider until you find out what materials you will have to work with.

Donna Hardy

[Trader]

Thanks for the advice regarding the emailing =)

Coool. Ooh, then if I get lucky, a sensor strain is there! This is worth trying. Good thing is, with bacteria, they grow fast =) and hopefully with that, results come back fast too! xD!!!!!! IF I get the hang of microbiology processes fast enough, is it possible to conduct two experiments at the same time such that I can attempt both the sensor strain idea and the AHL v. distance idea? -- If I can't, I think I think I might head towards this route! I'll really appreciate the help that your colleagues will give! Thank you very much for this

Regarding the AHL v. distance -- at first I thought it was somewhat impractical as people know that sensor strains should be grown close to the test strain anyways, and that the varying distance might not be of much importance, and that is if I manage to get the "maximum" distance travelable. But then if I think about it, it is something I really want to find out about, and it might give us more information about the structure about the signals, especially w/ b. subtilis which isn't studied as much =).

Now I have to write a research plan by tomorrow, and start experiments the day after. That's not going to happen...Aii, should have started sooner

Update

Wow...I started writing the research plan for the AHL v. distance idea, and it started being very appealing! My RQ will be something around:

Which is more diffusible through culture plate media when using sensor strains, e. coli or b. subtilis QS signals?

But I'm also going to see the "maximum distance" between them might be. I was also hoping that by finding out which signals are more diffusible, I might be able to explain some of their processes, such as why some bacteria release glucose [to make their signals more diffusible] or lower the pH so their signals are more diffusible.

Thank you so much :')

[Trader]

Some concept questions:

I've checked out https://www.sciencebuddies.org/science- ... ains.shtml and it's very useful, but by going through the short-term storing, that means that the bacteria are in a condition of minimal growth and after 1 month, would still be in the lag phase?

If the strains that I already receive are in a stationary phase, I can inoculate a "fresh" strain by having the bacteria "moved" into a fresh agar plate right?

(I don't think I can access the materials in the long-term processes)

For b. subtilis, ComX oligopeptide pheromones control "competition" factors in b. subtilis...

How may one "detect" the amount of competition there is in b. subtilis?

I researched everywhere for previous experiments with a "b. subtilis sensor strain" but there has been none -- is this because it is almost impossible to use b. subtilis as a sensor strain? -- as sensor strains must exhibit some visible effect such as bioluminesence or the release of violaceum?

And just to confirm (this is a question that I've searched everywhere for... I thought I would know the answer, but it seems like the articles I have access to automatically go to a very advanced experiment and doesn't really answer:

To confirm: E. coli does NOT produce AHL, produces AI-2 (any other signals? I wanted to make sure, but I didn't find any) and the ONLY produces AI-2, which controls e. coli cell division ONLY

This makes me want to get v. fischeri so much more! I'll see if I can access v. harveyi too , and in the meantime, see what I can do with b. subtilis and e. coli AI-2 detection.

If I can find out through my own experimentation, then I can. I'm not sure though, because there are ambiguities I have regarding the sensor strain method -- but is it possible to somehow find out what functions e. coli quorum sensing controls?

And...(sorry!! but I'm somewhat desperate. This wont happen next year.

To determine the amount of bacteria in agar plates (not liquid broth cultures), does dilution plating work? Sources say to pipette 0.1 ml of the culture into a dish and dilute it with 99.9 ml of (distilled water?) for example, but pipette is for liquid sources, which an agar plate isn't...so it possible to 'transfer' one bacteria colony into a liquid medium, and then dilute that?

I am very sorry for all this rush )=...especially with these concept questions -- I tried researching w/ articles, but with things like b. subtilis sensor strains, I can't find previous experiments on those (Is there a reason? Because I'm pretty sure even the popular vibrio sensor strains cannot detect protein peptide QS signals...) -- as for the rest, please understand I'm using this as a last resort .

Thank you for your patience in explaining everything

[donnahardy2]

Hi Trader,

It sounds like you a busy thinking about your project again. I'm hoping you will hear from one of the researchers who are involved in your topic within a couple of days. It will make a big difference if you know what you will have to work with. Here is some additional information:

Bacterial cultures: If you have bacterial cultures on agar slants or plates in stationary phase, you can transfer the culture to a new medium and grow it up again to make a fresh culture. In fact, you would want to do this frequently to keep your cultures viable while you are working on your project. After a month in culture, bacteria would be probably be in death phase, and you would definitely need to transfer it to keep it alive. Lag phase refers to the early part of the growth cycle, which is right after the culture has been transferred to fresh growth medium and before the cells start dividing. Does that answer your question? Or, did you have another concern?

Doing plate counts is one method for counting bacteria. A broth culture that is visibly cloudy with bacteria will contain more then 10 million organisms per ml, and the initial 0.1 to 99.9 ml gives a 1:1000 dilution for starting a plate count. If you have access to micropipets, you can also start with a 10 microliter sample diluted in 10 ml. Generally, you can count plates that contain between 30 and 300 colonies, so to make a count, you need to plate several dilutions, grow the plates overnight, and then multiply the number of colonies on a countable plate times the dilution factor to get the number of bacteria per ml of sample. This will make more sense when you actually start doing plate counts in the lab.

I think you are right about B. subtilis. This organism produces peptides, which can interfere with AHL function of the Gram-negative organisms, but you would need a Gram-negative sensor strain to detect the presence or interference of the AHL's. There's no point worrying about the specific sensor strain, however, until you can obtain one to work with. All of the sensor strains are models used to detect the presence of AHL's in a sample.

The area of E. coli (and other Gram-negative bacteria) quorum sensing is a relatively new area of research and I think that there is potential for you to discover something new about the effect of AHL's on this function. So far, in the literature, I have seen references for food spoilage, plant pathogenicity, and biofilm production. There are probably lots of other topics that are affected by quorum sensing. I think the topic of communication between bacterial species is particularly interesting.

If you don’t have a reply from someone by tomorrow, then go to the American Society for Microbiology website http://www.asm.org/Search/SENSearch.asp, and search for a local mentor. I did this for my state and received a list of 10 local contacts with e-mail addresses and phone numbers that are available to help high school students with projects in general and applied microbiology. The success of your project depends on finding a knowledgeable microbiologist who can help you with specific advice and with getting the sensor strains that you need for your project.

Donna Hardy

[donnahardy2]

Hi Trader,

I just noticed that I didn't respond to your question about AHL production by E. coli. Remember the AHL's have various carbon chain lengths and various AHL's are produced by different Gram-negative organisms. It's likely that wild type Gram negative organisms produce a variety of AHL's. The problem with E. coli is that there are lots of strains that have been genetically modified, including the AHL-negative one used as a biosensor strain. So, you have to know the characteristics of the particular strain of E. coli that you are working with. Here's a fun website that explains the mechanisms of the AHL's:

http://2008.igem.org/Team:Calgary_Wetwa ... ideFrame_1

It sounds like these students had fun doing this project.

Donna Hardy