Genetic bacteriology experiment?

[Answer Go Boom]

I'm a highschool student, and was wanting to do an advanced-level bacteriology project pretty similar to the one AP Bio students work where E. coli is genetically modified to be ampicillin resistant using the enzyme EcoRI.

 

My twist on it was that I would like to do an experiment implimenting the gene for bioluminescence (taken from a species of firefly) into either Staphyloccus aureus or E. coli. The reason why I picked these bacteria strains is because of availability (they exist very commonly).

 

As far as materials, I have two campus laboratories I can use and may be able to use the local college biolabs. I can also arrange to get blood agar plates for culturing from the local hospital lab, so those are not an issue.

 

But considering I've never performed anything of this caliber, I need to know a few things.

 

A) How readily available is the enzymes (would I need them?), and the gene for bioluminescence?

 

B) How much of the experiment could be performed in vitro?

 

C) How much culturing would be necessary?

 

D) How would removing and implementing the gene be done, in a nutshell?

[ecoli]

you probably should use GFP (green flouresent protein). It's a common "glowy" protein that is easily used in making recombinant plasmids.

 

All of the experiment could be performed in vivo, with basic culturing techniques that would be easy to learn.

 

What you need is an assay of genetic cloning (quite common on the web)

 

You won't need blood agar for E. coli.. simple LB Agar (and LB liquid cultures) will do the trick.

 

Reagents needed: GFP gene, host plasmid, Restriction enzymes, ligase, chemically competent host strain...

[Answer Go Boom]

Wow, thanks.

 

And where would I go about getting them? Cost?

 

Are they common enough to just Google?

[CharonY]

If you really want bioluminescence you would just clone the luc (luciferase) gene. However you would need an in vitro assay to really see that. Probably Ecoli suggestion is easier, as you only need to excitation light of the proper wavelength (around 390 ish nm).

 

a) you would not need the proteins themselves, you would only clone the genes. This is most easily done either by buying a plasmid with the genes already cloned in, or, if you want to clone yourself, you will need everything to do a PCR.

 

b) the whole cloning part is. The GFP or Luciferase assay can be done in vivo as well as in vitro.

 

c) it depends on the vector. Ideally you would have two markers (selection and counterselection) then you only cultivate them twice (once for selecting for vector and insert, once more for the actual test).

 

d) PCR to amplify gene of interest (requires DNA with gene of interest plus suitable primers), cut the vector (or use a TA vector and a polymerase that does T-overhangs) ligate vector and insert, transform cells with it, select for vector, select for insert.

[Answer Go Boom]

Wow, guys, thanks, you've given me a lot to chew on.

 

Which would be easier to modify, Staph aureus or E. coli?

[ecoli]

Wow, guys, thanks, you've given me a lot to chew on.

 

Which would be easier to modify, Staph aureus or E. coli?

Probably E. coli, but just because I know it's used more as a model organism. Not entirely sure why that is. Perhaps just anachronism?

[Answer Go Boom]

I almost thought it might be because of its abundance. Considering the bug lives in the G.I. tracts of mammals, well...uh, there's no shortage of it.

[ecoli]

yeah, but I don't recommend trying to culture E. coli from your own intestines.

[Answer Go Boom]

Yes, I certainly agree with that.

 

Must not be very pleasant. I perfer methods of bacteriology which will not interfere with my ability to walk.

 

The cultures high-end labs use is purified anyway. No exterior or biological contaminants.

[CharonY]

Uhm guys, you are aware that

S. aureus

are biosafety 2 pathogens? Not that something to play around with (without proper training at least).

 

The cultures high-end labs use is purified anyway. No exterior or biological contaminants.

I am not sure what you mean by that? Even a oure (clonal) culture of pathogens is pathogenic (more so actually).

 

Probably E. coli, but just because I know it's used more as a model organism. Not entirely sure why that is. Perhaps just anachronism?

Not totally. It has some historic reasons, but even then they were not randomly selected. Main reasons are the easy genetic access to to E. colis and fast growth combined with easy cultivation.

[Answer Go Boom]

A) I will be using versions of Staph cultured from the skin, assuming that I do use Staph and not E. coli. And, correct me if I'm wrong, but only methicillin and vancomycin-resistant Staph aureus are considered to be BSL2 level. Now, applying statistics shows that because MRSA and VRSA are still relatively rare, there will be not many parts of the Staph culture that are resistant, however, there will be some.

 

Needless to say, I'll play it safe and be careful.

 

B) By "no exterior or biologic contaminants", I mean there's no dirt or non-bacterial matter in the culturing.

[CharonY]

A) Any potentially infectious bacteria are at least BSL2. This includes all S. aureus strains, regardless of resistance. Moreover, if you try to isolate from human samples you can cultivate a host of potentially infectious bacteria. Again, do not do it without proper training. That is, it is well outside the scope of a highschool student. Just trying to amplify something from human skin is well outside of safe and careful.

 

B) The media is non-bacterial matter. And some media are literally dirt. Only sterilized dirt.