Sciencemadness Discussion Board

Plasmid vectors, E. coli, Bacterial transformation

crazyboy - 13-12-2010 at 21:26

Last year I took AP biology and one of the labs entaled bacterial transformation of a non-pathogenic variety of E. Coli. The plasmid vecotr contained the lacZ gene a sequence which codes for the tranwsformation of "X-gal" (bromo-chloro-indolyl galactopyranoside) to a blue compound (5,5'-dibromo-4,4'-dichloro-indigo) The vector also contained a gene for Ampicillin resistance which allowed easy screening. The procedure was remarkably easy and only required micropipettes, gloves, PDA agar, petri dishes, a water bath etc.

I would like to conduct similar experiments at home, does anyone have a reputable source that will sell plasmid vectors with a antibiotic resistance as well as other biotech tools? How viable is this goal?

Attached instructions for bacterial transformation as described
but inserting a different gene.



Attachment: BIO-A-06A-COMP-pglo_bacterial_transformation.pdf (205kB)
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phlogiston - 14-12-2010 at 16:32

It isn't hard. in a properly equipped lab it is a routine 10 minute job.

I would imagine the hard part if you want to do it in a non-professional setting will be to aquire some of the materials.
I don't think there are any legal requirements for owning/obtaining DNA. Many scientists will probably send you a plasmid upon request. I have many times requested and received plasmids that I needed for my own research from other scientists. E. coli is a common organism, although I would not know how to isolate it from scratch. In a real lab, you simply buy the exact strain that you need and propagate it as necessary. You are also going to need the antibiotic to select succesfully transfected cells. Ampicilin is very common. Other (slightly less commonly used) ones are chloramphenicol and kanamycin. You are also going to have to make your bacteria 'competent'. Competent bacteria take up foreign DNA much more efficiently. There are several procedures to accomplish this. Google it.

In many countries, however, there are legal requirements for how to work with genetically modified organisms. They should not end up in the environment. It is not difficult to do, however, unless you work with pathogenic organisms. Read up on how to handle them properly. E. coli is not particularly dangerous. Essentially, you should make sure to kill any bacteria you dispose of (using bleach, NaOH or autoclaving).

Random - 17-1-2011 at 15:29

What is the way of actually obtaining plasmids from some organism DNA? I read that DNA should be extracted and then mixed with chloroform and phenol to dissolve proteins and leave plasmids, but what is the actual procedure for genetically modifying organisms starting from cells - then DNA extraction - plasmid?

nitro-genes - 19-1-2011 at 13:52

Plasmid miniprep or alkaline lysis protocol:

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

It really doesn't use dangerous or difficult obtainable chemicals. Centrifuging is most easy, though you could also draw the lysis buffer + plasmid through a glass filter to which the plasmid DNA attaches. (Glasswool maybe?! :D) Wash glass filter a couple of times with some 70% Caribbean Rum or Cask strength wiskey and finally elute your DNA with (hot) water.

The real difficulty is preparing the competent cells, which are only competent when in their early growth fase. When making competent cells you need to put the culture on ice at a pretty exact optical density. (OD600 of 0.2 or something) The cells need to be spun down multiple times in a cooled centrifuge and treated with a variety of chemicals to make them chemically competent. (Inoui, Hanahan, CaCl2)
More easy is preparing electrocompetent cells, though you need expensive equipement to perform the actual transformation. Buying compentent cells is not possible and pretty expensive, not to mention you indeed need a licence to work with GGO's. :)

[Edited on 19-1-2011 by nitro-genes]

peach - 19-1-2011 at 16:43

Inserting plasmids is not tricky no, there are numerous methods to do and, like the PCR methods, they are not all that difficult.

As I think you're getting at, actually obtaining the correct plasmid is harder work. Because it usually means creating knockouts, screening them, finding the sequence that has altered and then deducing how long that gene is before splicing it out and forming the plasmid - which is sequencing.

Uni's are spoilt for choice, as the staff can open the bio catalogues and flick through for what they're after a lot of the time, and, they don't need to pay the hundreds of dollars for the tens of micrograms. Students are even more so, as they don't even need to open the catalogue, it's on the shelf and the method is in a handout.

You could try setting up accounts with the bio suppliers, which may be as difficult as the chemical suppliers or more so - as less people are interested in this at home so getting specific gene sequences is like picking fine research chemicals and expecting a photography store to have them.

Asking university staff is another option, but again, as with chemicals, I expect they will be very reluctant to give them to the public.

Random - 21-1-2011 at 15:16

I should try that, actually for the beginning I would just make some random plasmids from DNA and then mix them with E. coli. Could I genetically modify yeast cells like that also?

By the way, I see you are talking about gene sequences, splicing and all that. Can you recommend me some good book where I could learn that easily?

peach - 22-1-2011 at 07:56

Work on modifying, splicing and selecting sequences starts in the A-Levels in the UK (16-18), but it's only the basic idea of identifying cultures (as you've done with the blue staining and antibiotic resistance) and you don't actually do any of it in school.

First year university biology text books on genetics will have sections on it. But again, you don't actually do much (and I was doing molecular cell biology).

This is a huge topic that can get extremely complex.

Adding the plasmids is akin to doing an acid base reaction nowadays in chemistry when all the gear is on the bench for you and you're working from a handout.

Selective splicing and modification of DNA is more like doing that same neutralisation but using the earth, wind, fire and water periodic table. There is a big jump in the level of effort required.

I would start by questioning what it is you intend to produce. For the majority of simple molecules, you'll get a far easier, cheaper and purer result by using a chemistry lab. Genetically engineered strains are usually used when the product starts becoming too complex for a chemistry lab to do a good job of it.

There are so many potential problems involved in modification, for instance the product may be toxic to the organism, or the genes you implant may do nothing because they're a stage that relies on others being present.

Genetic modification is usually used as an extreme method. Before that, you'd look for species that can already produce the material, then plate them and look for high yield variants. By allowing them to reproduce sexually, you will get variation and can then clone the best. Going up a stage in effort, you can add mutagens to accelerate the variation rate.

It's also worth looking at modifying the growth media, conditions and adding precursors or promoters to the culture.

There is little point to going through the effort of splicing if you're not going to gain something substantial from it.
Quote:
The elemental system used in Medieval alchemy was developed by the Arabic alchemist, Jābir ibn Hayyān and others.[9] His original system consisted of the four classical elements found in the ancient Greek traditions (air, earth, fire and water), in addition to two philosophical elements: sulphur, ‘the stone which burns’, which characterized the principle of combustibility, and mercury, which contained the idealized principle of metallic properties.

phlogiston - 24-3-2011 at 15:47

Yes, you can also transform yeast with plasmids, but yeast cells are slightly harder to transfect and require different plasmids.

If you just want to transform E. coli with a random plasmid, I guess that is doable. -Making- a specific plasmid to your needs, however, is a lot harder. Not particularly difficult if you have the materials available, but much of what you need for it is going to be nearly impossible to obtain outside of a professional setting.

I don't really see the point of transforming a random plasmid other than the learning experience.

Chalo - 31-7-2017 at 08:44

Please look at this link:
https://www.carolina.com/biotechnology-teaching-resources/tr...

Carolina has everything you need to transform E. coli including some interesting plasmids. Their pgreen Gfp plasmid is really cheap at $12 for enough to do many transformations. They also include detailed protocols. There are other plasmids available but the ones used for sci education are best to start because very manageable and easy to visually verify transformation. By far the best most sophisticated educational kits are from biorad:
http://www.bio-rad.com/en-us/education

but they are not cheap. When you get to the next stage of editing DNA and making your own plasmids, there are detailed plans for maiking your own equipment or getting used equipment and supplies both here and in other biohacker sites. It is not cheap, but can be done for a few thousand instead of a few hundred K.

Finally, if you are located near NYC or San Francisco, you can join genspace or biocurious and use their equipment. Please google it. It will inspire you. Do not give up your dream, biohacking is on the rise.

NEMO-Chemistry - 17-11-2017 at 05:58

If your in the Uk, then these will sell some things to private people. Only has the basics though

http://www.betterequipped.co.uk/science-equipment/biotechnol...

If i come across better links i will post up