Science Buddies: "Ask an Expert"

I want to work with melittin, a component of bee venom that has been known to induce cell lysis. I've narrowed it down to two of the following topics:

A) Varying the amount of melittin and seeing the survival rate of bacteria (any suggestions as to what is a feasible bacteria I can test on?)

B) Adding melittin to algae and seeing its oil production rate

What is the better of these two projects? Which one, if any, would actually work? Lastly, do you have any other ideas of projects similar to this that I can conduct?

Hi,

From the timing of your question, it sounds like you are planning ahead and are interested in working on a long-term project. Both of your topics appear to be excellent and both have practical applications so would be relevant. You have obviously put a lot of thought into this project already, but I would recommend that you spend a little time to do more background reading and develop your topic further. For a potential prize-winning project, you should ready the current literature and find out what problems need to be solved and then do a project that is unique and that answers the next question on the topic.

Please let me know what you have learned about melittin so far. Why are you interested in this peptide? Also, how are you going to get melittin for your project? You will need to have a supply of the peptide and a method to measure its effect with an assay.

Here are some examples of research reports on melittin:

http://cosmos.ucdavis.edu/archives/2010 ... _Diane.pdf

http://www.cell.com/biophysj/retrieve/p ... 9504737157

http://sbms.ccmu.edu.cn/extra/col1/1235103605.pdf

Here’s a news report on cell lysis to improve oil extraction from algae; it would be interesting to see if there’s a scientific report with this information available also:

http://www.oilgae.com/blog/2011/02/dive ... ology.html

One important consideration in selecting a project is the resources you have available. Do you have access to a laboratory or will you be working at home? Also, which project seems most interesting to you?


Donna Hardy

Thank you so much for taking the time to answer my post. All of your links were truly helpful. As for choosing melittin, I did so because I have heard of its ability to kill cells. I believe that it does this by rupturing the membrane. Here is an interesting article about melittin that talks about this:

http://www.jstor.org/pss/4295647 (This shows only the first page, but I also have the full article if you need it)

As for purchasing it, I was planning on buying it from Sigma Chemical:
https://www.sigmaaldrich.com/catalog/Pr ... KEY&F=SPEC

Do you know how much I might need for either experiment?

I am still quite confused as to which experiment to conduct. I believe that the experiment involving bacteria will actually show results. However, it has been done before with the bacteria that causes lyme disease:
http://www.ncbi.nlm.nih.gov/pubmed/9233 ... stractPlus (I also have the full paper of this)

For the experiment with algae, I am not sure if there will be any results at all. I believe that oil extraction methods are aimed to rupture the cell membrane of algal cells. Is this true? Also, do you think that melittin will actually be able to kill the algal cells and release their oil?

I believe that I definitely do need to use the resources of a research lab. I live in the Maryland, Washington D.C., and Northern Virginia Area. Do you know of any labs in these areas that have the resources necessary and would be willing to help me?

I know this is a lot, and I truly appreciate your help!

Thanks again!

Hi,

Melittin is a very expensive peptide, and you might consider doing a project on improving the purification method. If you do purchase the peptide, you would want to plan your experiments very carefully and work on a very small scale. Purification of this peptide would be a complete and challenging project on its own.

A local mentor would be very helpful. Since you are interested in investigating the use of this peptide with microorganisms, go to the American Society website and click on the “find a mentor” link on the home page and search for someone in your local area.

http://www.asm.org/

Also, since you are reading the journal articles, look at the location of the authors and look for a local author that you could contact for assistance. You should also ask your science teacher for suggestions for local contacts that might have access to a laboratory.

For an experiment, an anti-microbial or anti-cancer experiment would be suitable. You would probably want to use a non-pathogenic organism like E. coli as a model because of the problems involved with working with pathogenic microbes. I don’t think the algae experiment would be as good because for biofuel production, cost is a primary consideration and you would not want to use an expensive peptide to solve the cell lysis problem because the high cost would make the solution impractical. You could work on the algae cell lysis problem, but your objective would be to find an easy, very inexpensive method. Cost is a lower priority when trying to cure an infectious disease or cancer.

While you are looking for a mentor, continue reading as many articles as you can find. Look at the materials and methods sections and think about an experimental design that you could do. You may not find a local laboratory to work in, so you will need a plan to do the project on your own as well.


Donna Hardy

What do you mean by the purification of the peptide?

What are the processes required to work with pathogenic bacteria? I feel that it would give my project a more realistic and dramatic appearence. As for anti-cancer, there has been work done on nanobees that target and kill cancer cells. Do you think that if I work with a different type of cancer that I might lose points on non-originality of the project?

Finally, do you know of any other cheaper enzymes that conduct cell lysis?

Thanks for all of your help!

Hi,

Here is the information for getting approval from the scientific review committee before starting your project:

https://www.sciencebuddies.org/science- ... _src.shtml

To work with pathogenic microorganisms, you would need to have access to a laboratory approved to handle the type of organism. You can do an original project using a non-pathogenic organism as a model for you idea, so keep all of your options open at this time.

You should also review the information on the Science Buddies website for doing an advanced project:

https://www.sciencebuddies.org/science- ... ndex.shtml

And here are some more ideas for finding a local mentor:

https://www.sciencebuddies.org/science- ... tors.shtml

For an original project, you will want to read the scientific literature on your topic and identify a problem that needs to be solved, and then design an original experiment to solve that problem. Progress in science is made one small step at a time, so the originality could be in looking at a new technique, a different line of cancer cell, a different way of delivering the cell lysis agent to the target, a different way of purifying the melittin, or something else. At this point, I recommend that you concentrate on finding a problem to solve that you would actually be able to do with your resources. Finding a local mentor and laboratory will expand your resources, but you would also be able to do an original project at your high school or at home, if necessary.

I have been thinking about the problem of your sample. You could use crude bee venom for early experiments since the primary component is melittin. If necessary, you could use purified melittin for a definitive experiment. Do you know anyone who keeps bees? Would your parents or neighbors object to having a beehive around for the summer so you could collect a supply of bee venom for your experiments? This is a good time to make a plan to obtain lower cost melittin.

http://www.youtube.com/watch?v=SGQso0dWwy8

Please let me know what techniques you are familiar with. Have you grown bacteria before? Do you know anything about cloning? What types of science projects have you done in the past?


Donna Hardy

Thanks for the tips!

I was wondering what you mean by purification. Do you mean the purification of the bee venom? Or the purification of the melittin?

Also, when do you think is the right time to contact someone and ask them to be my mentor? I am afraid that I might need to wait until next year to get this project checked. Or do you believe that I should get started on it right away and contact some professors?

I really like the idea of using raw bee venom, but I will not be able to keep a bee hive. Do you know of a reliable place where I could purchase bee venom? Finally do you think that bee venom will actually work in say the cell lysis of an algal cell or even the killing of a bacteria or cancer cell?

I have had some previous experience with large research projects. Just recently I have finished a project on the bioremediation rate of vegetable oil using Chlorella Vulgaris. This was a major research project that my team conducted over the course of the year. Unfortunately, I have had not any experience with bacteria or conducting assays.

Thanks again!

Hi,

You are welcome!

Purification refers to a using various techniques to separate the molecules you are interested in, melittin, from the crude bee venom. With peptides and proteins this is commonly done using chromatography techniques.

For example, here are a couple of abstracts for purification of melittin:

http://www.sciencedirect.com/science?_o ... archtype=a

http://ukpmc.ac.uk/abstract/MED/1682333 ... A52A6.jvm4

http://caocao.myipcn.org/science?_ob=Pu ... ba987a7117

You could do a completely original project by improving the method for purifying melittin.

For your project, you have two major limiting factors at this point. First, if you had a mentor and access to a laboratory, it would be possible to finalize the details of your project. For example, if you found a microbiologist with a BSL-2 lab, you would have access to everything you would need for a microbial project (autoclave, medium, incubators, hoods, etc). If you found a molecular biologist, you could do a cloning project. If you found a biochemist with a chromatography system, you could work on the purification. If you found an entomologist, you could focus on a project involving the bees and the venom. If you can’t find a mentor, then you will need to use your creativity and figure out what you could do at home or at school. So, I recommend that you try to find a mentor now if it is possible. You are in a prime area for research laboratories, so I think there should be someone available who could help you.

Second, you are going to need a good source of bee venom. Bee venom products are sold by health food stores, but you would need to verify the quality, and you would want pure bee venom, not mixed with other ingredients. Perhaps you could contact a local bee keeper and see if you could collect bee venom on your own. It would be a good experience for you and it might give you an idea for a project.


It sounds like you have good experience in doing a science project. Any of the possible projects here would involve learning new techniques for you, which would be good. You would want to avoid a project that would involve too many complicated new techniques.

Donna Hardy

Thought I'd point out that related anti-microbial peptides have been cloned and placed into various expression systems, which might provide an alternative source of peptide.

This group, for example, can produce a melittin hybrid peptide in yeast:
http://www.ncbi.nlm.nih.gov/pubmed/20111863

Here's a group that designed an expression system for use in E. coli:
http://www.ncbi.nlm.nih.gov/pubmed/18025552

Another group has a paper describing the cloning of bombolitin, a similar peptide found in bumblebee venom.
http://www.ncbi.nlm.nih.gov/pubmed/20350616

And a Russian group apparently has made transgenic plants expressing antimicrobial peptides in their leaves:
http://www.ncbi.nlm.nih.gov/pubmed/21434415

Papers like this can also expose you to approaches for purifying your peptide of interest from a complex mixture. Note that custom engineered expression systems like these give the user the option of adding "tag" sequences to the cloned peptide that make purification easier (although the purification columns used to grab the tag may be expensive."

Best,
Will

Hi thanks for all of your help!

I am still hoping to work in the area of microbiology. In recent days, I have become particularly interested in MRSA. However I see two main problems with my project:

1. I will need a BSL 2 lab, and apparently you have to be 16 or over to work in the lab (I am only 15). Also I wonder how many microbiologests will let someone this young deal with MRSA or other bacteria?

2. What are the procedures to getting my project approved, considering that I might be working with a dangerous bacteria and peptide? Do I need to get it approved by my school before I begin testing? If so, I cannot do any tests until next school year, so is it a good idea to still contact a researcher and request them to assist me?

Finally, do you know of any other cell lyctic peptides and/or other bacteria that someone would actually me to work with (and will make a signficant impact on my paper)

Thanks so much!

Hi,

It’s good to hear from you again and it’s good that you are still thinking about your science fair project.

It would be challenging to get approval to do a project on MSRA. You definitely would have to get approval in advance before you did any experiments with this organism. Here is the information on science fair rules with working pathogenic organisms:

https://www.sciencebuddies.org/science- ... ents.shtml

Here is information on getting approval for doing a project that involves hazardous microorganisms:

https://www.sciencebuddies.org/science- ... _src.shtml

Instead of working with MSRA, you could do your experiments with a non-pathogenenic organism like Staphylococcus epidermidis, a member of normal human skin flora.

http://en.wikipedia.org/wiki/Staphylococcus_epidermidis

This organism is the same species as MSRA so any results you obtained would potentially be significant for the pathogenic Staphylococcus, and there would be no problem getting approval to work with it since it’s nonpathogenic.

There are lots of bacteriolytic peptides:

http://bloodjournal.hematologylibrary.o ... 0.full.pdf

http://www.ncbi.nlm.nih.gov/pubmed/14975505

http://www.ncbi.nlm.nih.gov/pubmed/2501837

http://books.google.com/books?id=izTeyI ... es&f=false

This is an interesting abstract on an article about how Staphylococcus aureus evades natural bacteriolytic peptides and other components of the human innate immune system.

http://www.traveldoctoronline.net/staph ... E4MTU=.htm

Here’s an abstract from a book on bacteriolytic peptides.

http://books.google.com/books?id=n8SV9i ... es&f=false

I hope this will help you continue with your background reading and help narrow down your topic. Please let me know if you can’t access any of the references.


Donna Hardy

Just a quick question which will help me in narrowing down my project:

Generally, if i work with a researcher, would the lab pay for the supplies? Or would I have to pay for the peptides and cultures?

Thanks in advance!

Hi,

This is a good question and something you would need to discuss in advance with the researcher. One possibility is that you would be able to use general lab supplies like pipettes and microscope slides, but you might have to pay for specialized supplies needed for your project. It just depends on how much money the lab has available. If you need something expensive, you can contact companies that supply the item and request a donation. You would need to acknowledge the donation in your presentation. Have you made any progress in finding a lab to work in?

Donna Hardy

Hi,

Thanks for your answers! I've deteremined the general list of researchers that I want to contact, but before I do that, I want to come close to finalizing my project. Luckily after a lot of research, I am narrowing it down.

For the project, which do you think will be easier to work with: Cancer or Bacteria? This will determine who I will contact. Furthermore, what lab rules are there for working with cancer or bacteria, and toxic peptides? I am only 15 so I am wondering if I am even allowed in college research labs. Both cancer and bacteria sound interesting to me and I wouldn't mind working with either one of them.

Finally, for my IV, I was thinking of creating different mixtures using cationic peptides (melittin, berberine, and possibly ampicillin). Do you think that's a good IV?

Thanks!

Hi,

I think it would be a good idea to have a specific project in mind. Based on your background reading, what question are you trying to answer? What do you think would happen if you mixed the 3 peptides together? What would you use as a control?

You could work with cancer or bacteria, depending on the resources in the lab. Cancer research would probably involve working with tissue culture cells, which is very expensive, so working with bacteria would probably be the best choice. You should probably try to put together a proposal that you can show to the researcher and be prepared to demonstrate that you understand your topic. This would help convince the researcher to accommodate your request for lab space. You also need a back-up plan for a project you can do at home in case you can't find a lab to work in.

Donna Hardy

Hi!

So I contacted many researchers but it's been a month and a half and only one responded (with a no unfortunately).... Do you have any other suggestions as to how to find someone who could help conduct the project with me? Or is it time for me to start over?

I've also modified my research. Now I hope to use an alkaloid to eliminate biofilm (one of the main things which create antibiotic resistant bacteria) and then use a peptide to eliminate the remaining bacteria. This I believe would create a way to eliminate all of the bacteria and prevent almost all of them from growing resistant. I've also determined which alkaloids and peptides to use.

Thanks!