Bacteria [Communications]

[Trader]

Foreward

The lengthy "report" in part, is due my passion for digression -- I apologize in advance, though I confess I make that apology only halfheartedly .

Ultimately, the essential information would be in this color =).

Next I would like to add that I have browsed through the topics of genetics and plant biology under the various ideas provided, but unfortunately it seems like nothing "comes up"... )=

Finally I wish to say in advance that any help, even by patiently reading through this..."report" is very appreciated.

..................................Thank you all in advance

Background -- My "topic"

I've been inspired by paleontologists who uncovered the Burgess Shale and wonderfully enhanced their research through the fine preservation of trilobites, Marella, and the like, to uncovering fossils of anthropods and plants that reached great sizes in the Carboniferous period. It is wonderful to see the connections between species millennia away from each other and finding the connections between each fossil.

Then, when I first read about Darwin's Origin of Life and his attempt to create a pool of "life"...I can still remember his words to his friend Joseph Hooker: "If (and oh, what a big if) we could conceive in some warm little pond with all sorts of ammonia and phosphoric salts...[to create a pool of life]".

I knew then when I was given the opportunity to a science fair, that I'd pursue a topic in the area [of evolution and/or the origin of life]

Finding my research topic...

I have come up with several ideas, though they were only formed and I have, literally, no idea of the possibility of such an event happening (ahh, which reminds me when Marconi experimented with his radiotelegraph...but that's a different topic for another day ). When I went to brainstorm for topics, my mind immediately settled on the evolution of the peppered moth during the Industrial Revolution. Through research I have come across terms such as melanism, population genetics, and genetic drift, terms I am vaguely familiar with, but all which I have heard of. It appears that scientists have already attributed this evolution to natural selection and mutation, as opposed to genetic drift and migration; I appear to be at somewhat of a block in this area, so I wonder if there are any topics regarding this area which I could pursue? .

I have also looked towards the possibility of testing a certain plant in various conditions, and after the largest number of generations that time allows (I am looking to about 3 months of experimentation), conduct a bioinformatics report on, perhaps the DNA sequence of a certain gene in addition to observations of the differences evolution has carried out. I have conducted less research in this topic then the previous, though based on former years of biology science I believe the plant I am looking for would be the common pea plant (after all, the father of genetics Mendell experimented with them ). I could test different conditions to alter the allele frequency. I do wonder though, whether such a "report" which to me (though I state this on shaky ground, given the lack of research), seems like a "re-experimentation" of Mendell's work, would be appreciated by the science fair?

Given the past few days of brainstorming, I have narrowed possible research topics down to these two and I am constantly on the search for other topics regarding changes in evolution. Briefly looking back, I observe that I haven't had any ideas on topics more focused on the "origin of life". Simple concepts including "What makes something living?" and "What needs to be there for there to be life?" conjures flashbacks of elementary science -- which could accomplish the goal of having a research topic, but perhaps not a "competent", "worthwhile" experiment. That is not to say that I have given up yet however, as I've also thought about experiments regarding Rare Earth hypothesis and refining the Goldilocks Principle, though again I find myself wondering: am I doing what others have already done, or is my perception of the science fair's expectations somewhat distracted from my perception of what is expected by an inventor, for example?

Essentially, I am attempting to narrow down my ... two and a half research topics while factoring in 1) what is most "accomplishable", yet challenging? 2) what exactly can I do, having each topic somewhat narrowed down, but not refined?

[In regards to the "most accomplishable", I have wondered for a long time whether experiments on "evolution", a process that more often than not, does not occur over 3 months, is eligible as a science fair topic]

I shall continue researching to find out, but for now, would highly appreciate any pointers in a certain direction. Thank you =)

[deleted-71490]

You have selected a rather complex science fair project. I would suggest a Google search on short term experiments to demonstrate evolution or adaptation.

You may find working that with bactera or fungi will lead to faster rsults.

Good luck.

Matthew W. Mulanax, Ph.D

[Trader]

Thanks Matt --
Wow, that solves the question of achieving noticeable results in 3 months since bacteria do reproduce fast, hence known for their antibiotic resistance and other remarkable capabilities.

I could test for the differences in the DNA sequences after a certain type of bacteria undergoes a variety of environments. Would "Testing the effects of various environments on XX bacteria's DNA sequnce and evolution" be a possible research topic?

But again, I guess what I'm not sure is what "special" experiments I can do. I already understand the concepts of how bacteria develops antibiotic resistance, "shares" its genetic material such that it evolves to become a better strain to combat its environments...

[deleted-71490]

Understanding concept, in this case resistance development in microorganisms, is the most difficult part of planning and executing a research project . The next step is to formulate a simple experiment to demonstrate the concept then repeat the experiment with your test conditions - different antibiotics or environmental conditions (organism grows outside the normal pH range, normal temperature range or water activity level). I mention water activity because it plays an important role in microbial growth in foods and hence an important role in food safety. You might be able to demonstrate a change in an organisms ability to grow at lower than expected water activel levels.

The following sites wil provide some back ground information on water activity.

Search ResultsWater activity - Wikipedia, the free encyclopediaWater activity or aw is a measurement of the energy status of the water in a system. It is defined as the vapor pressure of water above a sample divided by ...
en.wikipedia.org/wiki/Water_activity - 39k

Water Activity Definition & ApplicationsWater activity is defined under static conditions of equilibrium. Under such conditions, the partial pressure of water vapor (p) at the surface of the ...
www.rotronic-usa.com/Ref/Aw/aw_define.htm - 12k

[PDF] Water Activity Definition: Water activity is a measure of how ...File Format: PDF/Adobe Acrobat - View as HTML
that it could not take part in a hydrolysis reaction the overall water activity would be. reduced. Water activity (a. w. ) is defined as ...
class.fst.ohio-state.edu/fst605/Laboratories/Lab%201_Water%20Activity.pdf

Water activityThe absolute limit of microbial growth is about aw = 0.6.b As the solute concentration ... The water activity (aw) is related to the chemical potential ( μw; ...
www.lsbu.ac.uk/water/activity.html

The following sites provide background on effect of water activity on microbial growth.

Microbial growth at reduced water activities: some...[J Appl ...No relationship of viscosity of the electrolytes to effectiveness as 'compatible ... 2000]; Strategies for microbial growth at reduced water activities. ...
www.ncbi.nlm.nih.gov/pubmed/6725160

Effect of the sugar-beet pulp water activity on the solid-state ...The growth and the sporulation of Tri-. choderma viride TS in relation to water activity .... tors at 28 °C for 3 days in experiments with microbial growth, ...
www.springerlink.com/index/T535J186071772G3.pdf

The sugarbeet pulp and water activity paper is in the journal Applied Microbiology (1987)26:537-541. Authors W. Garajek and P. Gervais. You might want to read this if your school librarian can get it or if you have access to a University library.

Good luck.

Matthew W. Mulanax, Ph.D.

[Trader]

Wow...thanks once again.

Interesting -- now I guess I have a redefined purpose in this project. This is just great, and the impact of water activity on bacteria is one area that I've actually been familiar with -- these sites are a great start, thank you.

Anyway, I will get started right away, this is, for the lack of a more "formal" word, AWESOME =). Thank you so much :')

With some outside inquiry, I think I might once again reconsider plants... it turns out there is a special kind of plant, "Wisconsin fast grass" if I recall correctly, though I might have confused the state with another one...with its fast 30-day generation cycle, I could get started perhaps 3-4 generations done -- perhaps that isn't enough for major changes in evolution, but I must admit that there is just something about plants in general that call out to me. But bacteria also calls out to me...as weird as that sounds .

Ahh, if only I had the time to do both. I guess I could conduct a side long-term experiment on plants for the next next science fair haha

[deleted-71490]

The “Wisconsin Fast Plants” are close relatives of cabbage and were selected for genetic studies. They are now used universally in K through post university graduate studies.

This site will provide you with information on ordering and use of Fast Plants.
http://www.fastplants.org/sandbox.paul.php#menu

Matthew W. Mulanax, Ph.D.

[Trader]

I've decided to research both topics until I've received enough information...

One thing ... if I start right now, I can get through approx. 3 life cycles of the Wisconsin fast plants. 3 cycles probably wouldn't be enough to bring out differences in genes right, or for any "interesting results" for a sci fair for that matter right?

Ahh, now I've started researching various topics. Every time I google a certain subject, there is always some kind of wikipedia page that appears.

I am very tempted to click on those, but based on your experience in high school international fairs, would using wikipedia pages as part of my background research lower my changes of advancing to higher-level or even Intel ISEF?

Thank you --

[deleted-2131]

Trader,

See my reply about Wikipedia on your other topic. Also, please note that we ask that you keep all your questions about your project on a single topic so that the Experts can better help you; doing so will benefit you and the Experts.

It sounds like you are off to a great start with an excellent projects that has the potential to go far. The advice you have ben receiving is excellent and I know that you will continue to get such outstanding help and mentorship.

Since you are intending to take this project to ISEF, you need to be following the international rules for precollege research. If you have not already, you need to talk to your teacher and/or the SRC of your regional ISEF-affiliated fair to make sure that you are in compliance with the rules and complete all of the appropriate paperwork. It would be sad if you went through all of this work but weren't able to go to ISEF because of a rules problem. Please check with your teacher and fair to make sure that you are in compliance.

Keep up the good work and good luck!

[Trader]

Cool, I'll get straight onto it. Yes, I've made similar mistakes in the past (though not for science fairs...but a 'rules problem', one of the worst of problems ) so I'll be sure to talk with my teacher. Thanks for the reminder!

For the brainstorming process in terms of finding the actual research question, I think I might dive straight into the complicated peer-reviewed articles <_<. They are very confusing, but I can find some sense here and there. This is usually how one narrows down their topic right -- through peer-reviewed articles and general background research?

Thank you once again.

[deleted-2131]

Trader,

Not everyone narrows down their question by looking at peer-reviewed articles, but those who go on to win big prizes at the top competitions (like ISEF) often do. If you're finding them difficult (which most high school students do), one strategy is to look up the words and phrases you don't know in Wikipedia or a similar source. As you start to understand those terms, it will get easier and easier to read and understand the articles. In fact, it will get easier with each article you read!

Keep up informed about how things are going and let us know if there is anything we can do to help.

[Trader]

xD It is getting easier, though I didn't do much research. I think I learned from the descriptions in the articles themselves.

I think I've narrowed another step down into the study of pathogens and virulence factors, host cells...especially after this interesting article on the P. gingivalis.

I haven't done much research in terms of the expenses that may be required...especially with things like bacteria, I'm not even sure I can "purchase" strains or something. In your knowledge, about how much is the cost of the average project in, say the ISEF? And I don't have a university-setting lab I have access to and I am considering pathogens... I only have a high school setting lab...D=.

Thanks

[Trader]

In other words, is it possible to research a topic that involves pathogens...by using "regular" bacteria as substitutes? (I think the answer would be no...)

Thanks

[deleted-2131]

Trader,

My apologies for the delayed response to your question. I think that there are valid points to be learned by studying non-pathogenic bacteria (depending on your high school and teachers, your HS may qualify as a BSL-2 laboratory; this would be something to talk to your teacher and regional fair SRC about). Many of the chemical pathways in bacteria are similar from one strain to another, while others are different. At this point, I think that one of the microbiologists on the Forums will be able to best help you choose a strain of bacteria that is both safe to work with in your lab and of scientific value and relevance to your question. (Post back with the BSL rating of your HS lab to help the Experts guide you toward appropriate strains.) I will ask that one of the microbiologists takes a look at your questions and gives some advice about how to proceed.

As for your other questions: (1) Yes, you can order specific strains of bacteria. (2) As far as expenses go...they vary. Some projects have much larger budgets than others, but that doesn't necessarily indicate the chance that a project has of doing well at the top competitions. Do think about the resources that you have, but also remember that the team of Experts on the Forums are VERY good at helping you find alternatives to more expense methods. So if you come up against something that is too expensive, let us know and we'll do our best to help you find an alternative.

Keep the questions coming! Hopefully a microbiologist will have a post here addressing your other questions in a few days.

Good luck on the project!

[Trader]

Oh no problem -- already, thanks a lot for helping us get through these blocks in our science fair project =P (and for spending the time to find microbiologists!)

I haven't had a chance to talk with my teacher yet, but based on what I've done there and some brief research of what a BSL 1 & 2 contains, I'm leaning towards a BSL 2...but I can confirm that as soon as the week starts again.

I've made another step (yay!), focusing on transportation/communication between bacteria. I've read many peer-reviewed articles (before I dreaded reading them...now they aren't so bad! =)), and I've noticed that bacteria rely heavily on their nutrient acquisition to secretion systems to communications (including horizontal gene transfers and all other transfers).

While I wish I can work with pathogens, I think it's best for me to perhaps base the project instead, on how to impede/control these processes [of nutrient acquisition, SSs, and communications] (I'll have to choose one) so that I can limit the damaging properties of a certain bacteria (instead of a pathogen =P), on a species of corn, for example. (I'm assuming by pathogens we mean more specific, human-harming strains or like mycobacteria...) This reminds me of the water activity research I've done (thanks for the websites again!), because that impacts nutrient acquisition...I'm currently researching more about communications to see which topic [of nutrient acquisition, SSs, and communications] I am most interested in. THIS IS SO FUN, and I wouldn't have made it ... well, this far (if it is far =P) without the experts' help. THANK YOU!

*--* Now that I look back, I see that I haven't made it much less vague, but it feels like a big step =)

*-- Edit # 2 --* One more question though; I'm currently using http://journals.asm.org/ "ASM Journals Online" and http://mic.sgmjournals.org/ "SGM Journals Online" as sources of my ... I think they count as peer-reviewed articles. But the free ones are running out...I've searched a lot for more free sources, but I'm not sure there are anymore...

One that thought, I just realized I could use Google Scholar... I shall see how that works out, though I've never used scholar before.

[deleted-2131]

Trader,

It sounds like you are making HUGE progress! Congratulations! It is exciting for us to see your project develop in this way. I am sorry that a microbiologist hasn't chimed in yet. I'll do some more poking to find one to help you out. I think your idea about focusing on nutrient transportation systems and mechanisms is very interesting, extremely relevant, and significant. It may seem vague to you, but to me it sounds like you've found some definite direction in your project and can start developing your methodology.

The sites you've listed are excellent sources of peer-reviewed articles. And you're right, it's hard to find ones that are free to access. Google Scholar can be helpful, but sometimes it will link you to articles that aren't free. One resource you might look at is PubMed: http://www.ncbi.nlm.nih.gov/pubmed/.

Although this preprint server is strongly weighted towards the physical sciences, there is a section on quantitative biology that might have relevant articles: http://arxiv.org/. Also see if some of these contain useful material: http://www.lib.uchicago.edu/e/su/sci/preprints.html. Also http://genomebiology.com/preprint/. AN IMPORTANT NOTE ABOUT PREPRINTS!!! Many preprints HAVE NOT been peer-reviewed. However, they can be useful because they are very, very current. This page contains some helpful info about how to user preprints: http://www.csulb.edu/library/subj/scientific_preprints/.

Incidentally, you might find this article that was recently added to the Science Buddies website helpful: https://www.sciencebuddies.org/science- ... aper.shtml.

I'll continue to try to hunt down a microbiologist...Keep up the excellent work you are doing! And keep us posted on how we can help.

Good luck!

[deleted-2131]

Trader,

I also forgot to say that a good way to get access to those articles that are not free is to visit the library of a local college or university. The school will usually have subscriptions to various databases of articles and if you are in the library, you can usually use them. It you have problems or questions, talk to someone who works at the library. There is usually a subject matter librarian (e.g. a librarian over biology) and they are typically very helpful at helping you find material.

[Trader]

Wow, I just checked out those sources and they work out great! Thank you --.

They are getting easier already, and now I have focused purely on bacteria processes of [NA/SSs/C], primarily on communications. The peer-reviewed articles are getting easier, and the interest levels (after I finish each article, I reflect upon it in my journal and write a rating out of 10 for each article so I can pick the top ratings and focus my topic in) have been getting up after each one! It seems like the more I understand an article, the higher its 'interest level'... I have thought about whether or not my first peer-reviewed articles were accurately rated, but then again, genomics and phylogenetic diversity doesn't appeal to me =P. I am focusing on bacteria communications, though it is also the last subject I've read about after nutrient acquisition and secretion systems. But then again, I find myself often distracted in the pursuit of knowledge and communications do seem to appeal to me more, so I think I have made another step in narrowing my topic down! As a side note...quorum sensing as well as biofilm formation and their relation to communications (as well as how bacteria can sense the density of their surroundings) are the top 3 aspects of communications that appeals to me... Hmm

>>>> How to impede/control a [certain area X] of bacterial communications so that I can limit the [X] effects of bacteria [X] on [X] <<<

Tada =). Ahh... It's 11/4 now...4 days after my original plan for 11/1 having a research hypothesis developed. I admit that there has been a lot of homework, but I see a break coming up, and I will now make sure that I have all the [X]s above removed by 11/14, in 10 days. Fortunately, by solving one [X] I solve all the others, though in another way it makes the solving much more harder. ...

Also thank you a lot for this article [https://www.sciencebuddies.org/science- ... aper.shtml], and amazingly and thankfully I am already doing many of the things. It turns out that I've been reading review articles (unless by peer-REVIEWED they are all review articles...), and the process is definitely much easier now.

xD Thank you! I love this site...it's like my e-lab-book. ** On a side note, I don't have a question yet, and I just like posting to mark my progress. =)**

[deleted-2131]

I just love your enthusiasm! It's contagious and it will take you far. I do agree that it is time to start narrowing things down to a specific project; you need to start getting your methods worked out so that you have time to get as much data as possible (the more data, the better) before the fair.

Keep us posted!

[Trader]

Ahh...I am now sweeping through peer-reviewed articles faster than ever, it's almost addicting.

I've decided to focus on something regarding quorum sensing and the bacteria's ability to cope with the density of bacteria in their surroundings. Though before I started talking about specifics, my high school teacher recommended that I start with something simpler, such as understanding the basic concepts of why any strain of bacteria, when grown in agar for example, would just stop growing after the colonies reached the edge.

Though I think it would be better for me to do research on the area, because I'm sure people have already detected the "why"...for me, it would be interesting to find out "how" this process can be controlled or altered.

As for the deadline, I feel that I am lacking discipline SO I will have a research question before I turn 16 in a few days.

*-- Edit. Ahh, previously my peer-reviewed journals weren't doing much narrowing down, as my RQ is now:

"the effect of X on bacteria X's quorum sensing abilities to do X [which will prevent/help bacteria X from doing X on environmental factor X]"

A small question though... I feel that I've worked "backwards" and away from reaching a final RQ...I would be fine with a research question [RQ] on the effects of X on quorum sensing alone, but I feel that I am lacking direction in where, and I am hoping to find an environmental problem where I can see bacteria in work, and see why it works, and even possibly how it can be prevented. Am I working backwards? O=

Now I've found http://aem.asm.org, a branch of the "asm.org" previously. Though while I focused on bacteriology previously, it seems that most of the articles under this AEM or Applied and Environmental Microbiology is dealing with bacteria, and I only have to avoid the few articles under fungi, viruses, and the whole section on mycology.

But I've found a whole new source of peer-reviewed articles on something more relevant - microbial ecology! XD

[deleted-2131]

Trader,

I don't think that you're working backwards; I think that you are refining your question and focusing your intent. I think that it may be more significant to actually start with the bacteria and understand their behavior and then take that information and apply it to ecological problems than to start with the ecological problem itself. If you can have a solid set of science, making a contribution to the microbiology community, and then take that science and apply it to the ecological sciences, you will have a significant and attention-grabbing project indeed.

I'm looking forward to seeing your research question! Keep up the good work!

[Trader]

Note: Text not in the quote boxes are just part of an online journal I'd like to keep =) if you don't mind.

Bleh...)= 2 weeks passed yet again, I am going closer and closer to the fair, yet I have accomplished little. )=

It could already be too late, but I shall continue nevertheless because it is just very interesting ... and perhaps continue it to be a two-year experiment. The plan is...that I get some sort of experiment going by the end of this week...But then again, I planned to do that more than one month ago ><

I think that it may be more significant to actually start with the bacteria and understand their behavior and then take that information and apply it to ecological problems than to start with the ecological problem itself. If you can have a solid set of science, making a contribution to the microbiology community, and then take that science and apply it to the ecological sciences, you will have a significant and attention-grabbing project indeed. //quote

Cool! I think it might be a bit challenging for me to start with the ecological problem itself -- so now I've at least managed to start somewhere =). Thanks!! (I hope I haven't started too late

- I've gathered a brief materials list for what to do for detecting A-HL signals in the typical G- bacteria ... (via high-performance or regular TCL), and I'll see how that works for myself...probably with a strain such as E. coli.

For a list of materials for my first experiment (see PRELIMINARY research question below), I have [prepared to ask my high school teacher tomorrow on possible sources] --
- Some strain of E. coli
- Strips and materials used in TLC detection of A-HL signals
- Agar plates for growing the bacteria
- Would I need any other supplies to create a "normal" setting which people "normally" test their bacteria under?

I would also like to test out other possible ways to detect A-HL signals...yet I'm not sure if I know of any other ways to detect them other than through thin-layer chromotography... ...After I learn from this experiment, hopefully I'll have a second step to go forwards to. Ultimately I want to know how to control these signals..., and best case scenario [if not too late], apply it to the real world =) [though we would all be doing this naturally].

- Then I will attempt to find what are 1) key ways that are being used to inhibit this communication in the world today and 2) how those ways can be improved or what other ways there are.
- THEN I'll attempt to still find that bacteria X, and apply it there.

It is a rather simplified version, but hmm...

I can't seem to find what "bacteria-related" problems there are with the world today...that is not related with human diseases =P. If I can't find any, I'll still begin the experiments learning how to inhibit these signals so I'll see what I can do from there.

I've briefly looked at plants, but bacteria infecting plants have the proper bacterial treatment...

So I am thinking at bacteria at work, perhaps through a process that negatively affects us...

OR I can attempt to promote an important process such as the nitrogen cycle through enhancing its communications...though that might not work out because the bacteria that is in the soil is probably in its optimal condition, and if not, it would be impractical to change it...

But then again...I don't think I would immediately find a bacteria process or "bacteria problem" in the world =P. I am just brainstorming now (and I realize that this was supposed to be done a LONG time ago...but...ahh no excuses )=), I could...improve some sort of way to detect those signals, or find the optimal conditions for bacteria to grow...That would be interesting.

For some bacteria experts out there > my 'preliminary' research question (I guess we can call it that) is: How effective is the use of thin layer chromotography and other methods in detecting A-HL signals used in E.coli quorum sensing?

I'm not sure if that's a good start...but what I'm really trying to do here is:
1) Get some preliminary information and experience so I can expand on that and see what other research questions might pop up though
2) I'm not sure if a "how" question is a good way to start...and
3) For the "and other methods", what I'm really trying to do is find a way to a) detect these signals and b) control these signals, without having to ...use the super fancy machines I've learned about =P such as HPLC and even nuclear magnetic resonance machines O=

Oh )= ... but I'm not out of hope yet =). I'm sorry, I think the little that was accomplished in 2 WEEKS AFTER when I was supposed to start the experiment... is disappointing for both me and everybody around )=

Some good news though...I am definitely going to start gathering the supplies tomorrow =).

I really love science...but sometimes life sets these challenges (i.e. homework)... =P but I've come this far, and I know my interest and the fun that'll come out of it will be worth it...[if it's not too late]

---!!UPDATE!!-- > New Day -- asked my high school science teacher =)

Now I have new goals:

1) Find out exactly what I need to look for in thin layer chromatography and
2) start locating a non-pathogenic bacteria strain...it's all very interesting!

Even though it's exams time, I find myself having more time to actually dedicate myself to this project now! I hope it's not too late! ><

Current plan: To find out what I should look for with TLC method of ID'ing A-HL signals, as well as finding alternate methods soon

[Trader]

Update: Dec 12, 2008

It looks like I can finally focus on one type of peer-reviewed article! On "detecting and inhibiting AI-2/A-HL signals used in quorum sensing in vibrio bacteria (a type of bacteria that uses AI-2 signals)"

>< Finally I have a purpose, though it looks like there aren't a lot of labs out there that can show me other ways to detect these signals, that are "achievable", that is not with the use of thin-layer chromatography.

But then again, I have yet to read through my finds =), but for the biologists out there,

Am I heading in the right direction by attempting to detect these signals? Perhaps I should do an analysis on which one is more effective later...

Thanks =) and I can't wait to finish up my research and actually get started!

[deleted-2131]

Hi Trader,

It is so exciting that you are finally getting started! You have put so much effort into getting this far;I know that if you continue with this level of dedication that you will be on the path to success.

We have someone who is familiar with thin layer chromatography who is going to look at what you're proposing and provide some feedback. She is really busy right now, and so probably won't be able to get some feedback to you until the end of next week or beginning of the following week.

Keep us posted and keep up the good work!

[Trader]

Thank you for the optimistic note...I feel like it's not too late! -- I can't wait! =) ** I'll edit this when I've gathered more research.

xD starting my own materials list >> this is so fun! xD

One question though...

Is it okay if a research question begins with 'How effective'? Because my research question is "How effective is the use of agar-plated bioassays and TLC to detect A-HL signals used in V. fescheri quorum sensing" ... Then again...I can't really construct a hypothesis >< ... unless science fair experiments are somewhat "different" and do not take the form of independent lab reports? o=

Thank you so much Terik Daly for all the help you've given to get me this far! It's been so fun!! =)
(I also recently saw the thread saying that Science Buddies needed more volunteers, so I was inspired to help maybe elementary kids the Experts on this forum helped the rest of us =) -- don't know if I'll get accepted, but exploring more areas of science has always been something I loved!!)

Thanks

[deleted-2131]

Hi Trader,

Nope, it's definitely not too late.

I think that "How effective" is a fine way to state the question for a project that will be as complex as yours, especially because you are in large part evaluating the efficacy of a particular methodology in performing a specific task. You might find, for instance, that TLC is more effective than agar plated bioassays (or vis versa). You would probably also be VERY interested in comparing the efficacy of these two methods to other existing methodologies. Your conclusions might hypothetically take the form of something like "TLC is extremely effective in detecting....blah, blah, blah..... Furthermore, it is more effective than ________, a current technique used to detect quorum sensing signals." And a "How effective" form is, contrary to what you said, perfectly conducive for use as question on which to base your hypothesis, particularly if you can quantitatively measure "effectiveness". I'm not familiar enough with the field to know potential ways to do so, but I am positive that with your extensive background in the area that you do. So your hypothesis might take the form of something like "I think that (some method(s)) will be more effective in detecting quorum sensing signals than (some other method(s)) as measured by (some measure of how you are detecting the signals). In addition I think that these methods will be more effective/less effective than (some current existing methodologies)." Does that make sense? In other words, no, a science fair project doesn't differ from a lab report in that science fair projects don't have questions and hypotheses; in fact it is probably much more important to have a thoughtfully constructed and well worded question and hypothesis in a science fair project than it is to have in a lab report. The difference is that the type of question and hypothesis you will be using for your project are so different from the kinds of questions/hypotheses you have stated earlier in your science "career". This is in large part due to the fact the questions you are asking now are significantly more complex than any question you might ask in a lab report.

Keep up the good work. It sounds like you're making significant progress. In fact, you have already (1) identified a topic of interest, (2) conducting extensive background research to familiarize yourself with the field and current questions, (3) stated your question, (4) begun constructing your hypothesis, and (5) started to develop your materials and procedures. That's quite a significant amount of work!

[Trader]

Wow! Thank you very much I thought I was getting a bit off with the hypothesis, but now it seems like it's not a bad idea after all =P. Hehe, there are so many questions to find out about. I wonder which is more effective, and most importantly why ... xD. Thanks for the confidence boost at the end ... as for it being a "current question", I double checked and I'm not sure what to make of this:


They'll be a lot of stuff coming up, so only the important things will be in this color...otherwise ignore =P (because the rest is to help me think and process )

In this article, it says that

Several methods to detect the presence of AHL have been described. AHL can be extracted from liquid cultures, purified to homogeneity by semipreparative high-performance liquid chromotography (HPLC) and ID'd by mass spectrometry and 1H nuclear magnetic resonance (NMR) spectroscopy. ...[Explains another method using bacterial sensor systems]....[Explains how TLC can be used] ...although these methods are very useful and highly sensitive, they do not allow for detection at the single-cell level or at the local environment. ... Furthermore, investigation of AHL expression based on population level analysis does not give information about local concentrations. A live bacterial AHL sensor that signals the presence of AHL molecules by expressing a reporter such as GFP can fulfill these requirements"

It would still be a "current" question for me to analyze the methods mentioned above, in a non single-cell level, non local-environment culture right? O=

Hopefully it's a current question... <:o

I've Googled many peer-reviewed articles regarding this subject, and this is as close as it gets...so I can safely assume that there has been no experiments conducted to test out the effectiveness, and for the case above, the experiment centers on using gfp-Based A-HL sensor systems, as opposed to testing the effectiveness...I hope I'm not rationalizing in vain ><

I've done a bit more research and it appears that an agar-plate bioassay detects the presence of A-HL while TLC specifically differentiates between the type of A-HL that is present, so perhaps I may not be able to compare the two. However, the good news is (not that the previous is bad news), I've discovered that I can somehow use reporter strains and a certain "gfp-based sensor system" to detect A-HLs too, though I still will need to do more research on that.

Update >> I've uncovered several similar experiments, with the a lab report, and a research paper all mentioned this "gfp-based sensor system" and I've done a bit of research ... it turns out that gfp = green flourescent protein (something probably to do with bioluminescence, which is associated with the OHHL used by v. fischeri during quorum sensing) ...

There's one concept I'm not sure I understand too well, and I don't seem to be able to find the answer...

With the image of a gfp I'm looking at, and the mentions of "activating" the lux operon (to somehow change the quorum sensing), I was wondering whether...

The creation of a gfp-based biosensor would require very 'high-tech' materials? I've attempted googling this, but it appears that I've only been traced back to the peer reviewed articles already have (perhaps it's a "rare" term? o=). Looking at the peer-reviewed articles, it appears to say that the construction of a GFP-based AHL sensor "was chosen as the molecular component governing control over expression of the GFP reporters" >> with terms incl. "expression" and "reporters", it looks like I'll need to alter the genes...which I'm pretty sure I cannot do because I don't have access to a college lab )=...so

Is there an "easier" way to create a gfp-based biosensor?"

There are many things that I did manage to find via googling, and it looks like what I am looking at is definitely TLC, perhaps an agar-plate bioassay (though I still need to research on how it works), as well as something by the name of an "AHL dose-response".

There is also the use of "mass spectrometry" mentioned in the source below, but with some research it appears that the materials required for that is even harder to obtain -- is this somewhat correct? I guess I want to keep it somewhat simple, though not let both my need for "advanced material" be an obstacle while at the same time, attempting to keep ti as simple as possible.

Many of these "types of detection systems" are obtained from the peer-reviewed article from the Applied and Environmental Microbiology Journal, titled "gfp-Based N-Acyl Homoserine-Lactone Sensor Systems for Detection of Bacterial Communication"

>< I just hope I can ... build one of those biosensors with the pretty much limited materials I have access to. Though my high school teacher says to not stop because I can't find something, but find a way to find it...hopefully it won't be too hard =P


As for a qualitative/quantitative way to measure "effective" -- tehehe I've come across some interesting strains, with the top one still as v. fischeri, then by e.coli and c. violaceum.

I'll plan to do a bit more research on each, but it looks like with v. fischeri, I am hoping to find out how close the experimental value (using each method) is to the "theoretical value" found from the bioluminesence (which is directly controlled by OHHL, an A-HL used in quorum sensing"...xD! Or I can determine the theoretical value in c. violaceum with the "purple spots" that is its specific indicator of the presence of A-HL)

Still a lot more research to do

Thank you for helping me get this far! I can't wait to see if I'm on any right track once the microbiologist comes ><

.

[Trader]

Update: We have an autoclave! xD ... Now I can make my own LB media. Also, it looks like that we don't have access to e. coli but to another strain, so as soon as I get the name of that strain...

I'm a bit more familiar with the use of TLC now, and I'll do more research with all the other types of ways to detect signals. Two recent topics kind of caught my attention and interest -- one is, since it appears that the methods of detecting bacteria signals are so different, it might be clear which one is more effective than the other, so perhaps I could aim on one and attempt to improve it? which may link to 2) I'll attempt to find out under which conditions is the use of TLC most effective (that is AFTER I've finished doing other research and seeing whether or not it is clear that one is more effective than the other) and I've read a recent though somewhat unrelated article testing the effects of CO2 and O2 on bacteria growth...it seems so interesting! I wonder if it's too late to "switch gears", though much of my research is still based on detecting the actual signals, and not on comparing each method yet...

I'll get back to you -- in deep thought right now =P

[donnahardy2]

Hi Trader,

Greetings. Terik asked me to check in and try to help answer your questions. I must say that I am completely impressed by your background research, and thoughtfulness in developing your research topic. I want to provide any encouragement and advice you might need in proceeding. I have just had time to briefly read though the postings on this topic, and I gather that you will be comparing a bioassay and TLC for detecting A-HL? Is this correct? This is a new topic for me, so I have printed out some articles, which I will read tonight, and hopefully have some additional comments by tomorrow.

Which bacterium are you going to use? E. coli and C. violaceum both grow well on LB medium. C. violaceum, for some reason, dies off quickly when kept in culture, so you will need to plan to transfer it to fresh media about once a week to keep is alive. E. coli will survive on slant tubes in the refrigerator for several weeks as long it doesn't dry out. I've never grown V. fischeri, but it requires a special seawater medium, and is apparently more difficult to culture.

Do you have your list of materials and a preliminary detailed protocol written yet? I'm sure you are aware of the time limitations on a science fair project, and I assure you that you will be wishing for time for one additional experiment at deadline, so you do need to proceed with the lab work as soon as you can. Have you located a source for the silica for the TLC, and do you have the glass plates? And, do you have a standard for the A-HL. Do let me know if you help in locating a source for supplies.

I'm looking forward to hearing about progress on this project! Do you have any questions that need to be answered immediately?

Donna Hardy

[Trader]

Hi! Thanks for helping out -- I apologize for the large amount of questions that are about to come...I shouldn't have waited until last minute to prepare everything and perhaps not set homework as a solid first priority previously...

I’m facing a bit of a problem in terms of which road to take in a “crossroad” I’m facing. According to the research I have done, it looks like the effectiveness of each method is already very clear, with clear “hierarchies” in terms of accuracy, listing a gfp-biosensor as most accurate, a bioassay as second, and perhaps the TLC as the 3rd.

Also, given some research, it looks like a bioluminence bioassay, for example, detects the presence of A-HL, while TLCs specifically differentiate between the types of A-HL (at least I think this is what my research is trying to say) I’m finishing up my research now, but I’d like to ask:

Since the two methods appear to not compare exactly the same thing, would that be considered another variable? (and therefore it wouldn’t be logical to compare them?) … Also, I’ve done an “originality” check on my experiment – it seems like nobody else has done the comparison before, but I have seen peer-reviewed articles mention the various ways which another method, using an fgp-based biosensor, is more effective than the TLC // I’m not sure what to make of this yet, but I’ll let you know within a few hours )

As a result,

I am not sure how I can expand on these “accepted” measures of accuracy for the above three types. I was thinking about either 1) continuing on with the topic in ways such as measuring the limitations of each type [as signals include detecting the signals, differentiating between the signals, and detecting/differentiating them in different environments] OR 2) somewhat continue on the topic by attempting to improve a method [I am thinking about the TLC, as it is most accessible]
I have done the preliminary research and know that the materials to prepare a culture (including making the LB, the bacteria).

As for the materials, it looks like we have access to e. coli -- interestingly enough, we have access to genetically modified e. coli with bioluminesence like vibrio strains.

We are also looking to see if we have vibrio bacteria...

Yes, it looks like C. violaceum is out, because in terms of the culture medium I have access to, it’s really only LB.
Here’s my current list of materials:

For the LB media (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 [I am checking if I have access to v. fischeri -- if I don't, my backup is e. coli]
  - Cotton swabs for inoculation

Materials for the TLC
- Microscopic slides (for the plates)
- Silica (solid absorbent) >> I have yet to ask my teacher if we have these materials, will return with answer within few hours.
- Erlenmeyer flask (for preparation of silica) - 500 ml
- Ethyl acetate (I’ve seen this being used in many experiments with the TLC, but I am wondering where this would apply?)

Materials for the bioluminesence bioassay
- A CDD Camera (?) for capturing the bioluminesence...
- Not sure yet, more research needed.

In terms of what I know about the TLC, I do have previous experiments with TLC, but I do not know what I would be looking for in the TLC plates as it applies to this experiment (hopefully with me finishing reading about the next experiments, I can know. One interesting thing did appear though:

I notice there are experiments that use liquid cultures, and another that use ... solid cultures (perhaps actual agar plates with LB non-liquid media) > in your experience, which one would be more "efficient" in terms of money, preparation time, and accuracy?

Most of the experiments included a similar process as below (this is from here: )

"The V. scophthalmi strains A089 and A102 were grown overnight in different growth media (mLB, mTSB, mPW and AB) and the supernatant was analyzed for the presence of AHL-like molecules."

I'm not sure, but is it true that

the analyzing of supernatants is only applicable to liquid cultures? (Since I believe supernatants refers to what remains in a solution after centrifugation...

I'm not 100% sure about whether I'll continue down this road yet, however, I am planning to at least get started by seeing how the TLC works, and possible ways to improve. IF it is not clear that the bioassay/gfp-based biosensor/TLC is more effective than the other two, then I'll continue down this pathway. Though,

may I ask whether these methods are in fact, "comparable" or would it be like comparing an electronic scale to a triple beam balance?

Hopefully the above won't be an issue, as the materials I'm gathering right now can apply to both possibilities (possibility 1) of comparing the methods and 2) of improving a method.

I also have another question though – since I am attempting to detect the signals, I know the composition of the signals I am looking for is OHHL or 3-oxo-C6-HSL (I believe this is the standard for the A-HL...but I'm not sure what it is O=), but

in terms of “detecting” the actual signal on the TLC, what would I be looking for?

(My teacher also asked me to identify what exactly is the composition of it, and I’m afraid this is a basic concept that is not mentioned in the advanced peer-review articles because it’s very basic =P)

Yes – I did start a bit late <_<. I may have spent too much time brainstorming for a “good” idea, but the good news is, results from bacteria come back fast .

Update: According to my research, it looks like there are different kinds of bioassay, including a sensor strain bioassay to a TLC bioassay, to a bioluminescene bioassay, all three ideas seem pretty interesting.

In your experience, are the above three methods (of using a sensor strain, using a TLC bioassay and using a bioluminesence assay) "different" enough to compare their effectiveness?

And another update: I am filling out the safety/risk assessment/approval forms right now, so hopefully I can get experiments started soon.

Some bad news though: I have winter break coming up, which means that I won't have access to materials -- also, my high school science teacher is really nice, helping out with the gathering of the materials -- I'm afraid I won't really be able to start for another two weeks. >> Is it too late then? (that would leave me with about 2 months of experimentation... 2.5 months at most...)

My plan is to have a research plan developed in these two weeks so that I won't have to stop between experiments, but in an ideal experiment, I learn from my results so that I can shape the future experiments from there. But I do want to apply for the fair this year...(though I plan on making it a 2 year project later) ...

Next time, the TOP thing on my list: START EARLY so I don't have to rush now!

A BIG thank you! (Again, SORRY for the HUGE amount of questions!)

[deleted-2131]

Hi Trader,

Lots of questions (and they are good questions) are often a key to finding meaningful information, so don't be worried about asking questions. Donna Hardy will be better able to address the science questions you raised, but I would say that if you have everything ready to go when you get back from your winter break, you should have time to get some meaningful data and analyze it before the fair. You just need to be ready and prepared so that you can hit the ground running after the break (and it sounds like you are on your way there). I'm also pleased to hear that you are envisioning this as a multi-year project; I think that that perspective will help you prioritize what needs to be done for this year; you need to identify your specific question to focus on for THIS year; the other questions will need to wait to be formally answered until your first experiment(s) are done.

Keep up the good work!