Well, first off, the AP skipped ahead a bit by describing the end goal of this particular project; they probably didn't have the space to elaborate on the proximate goal of transforming L. acidophilus with anything, which is the stage I'm at right now. Transforming Gram-positive bacteria is quite a bit more challenging than transforming E. coli. But, that said ...
What's your protocol?
For heat shock (which hasn't worked so far) I've tried a protocol from a patent that specifically focuses on lactic acid bacteria; it's a modification of an established protocol using PEG-8000, and I'll dig up the resource if you want to read it.
The heat shock protocol was pretty complicated, so recently I've been trying sonication, as described for E. coli here (http://nar.oxfordjournals.org/cgi/content/full/35/19/e129). No luck so far, though I'm going to do some more runs with better controls, and I'll try varying other aspects too -- so far I've only modified time, but I can also try temperature, CaCl2 concentration, adding some PEG, who knows; I need to do some more reviewing of the literature to get ideas, and have been keeping my eye out for a friendly microbiologist who works with Gram-positive bugs.
I would of course prefer to use electroporation, which was my old boss's suggestion, but electroporators are expensive. In addition to the wet-lab stuff I've been doing, I've also been working on a DIY electroporator; the logic side works, been having some problems getting the HV side to light up but that's what electrical-engineer friends are for. When I do it, I'll follow the protocol in "Optimization of technical conditions for the transformation of Lactobacillus acidophilus strains by electroporation", Kim, Han, Oh, You and Kim, J. Appl. Microbiology 2005.
Which plasmid/vector system?
At present, since I'm at the "get any old plasmid in there" stage, I have pGREEN plasmids from Carolina Biological.
Naturally, when we start trying to bang melamine deaminase in there, we'll need to build a custom plasmid for that. There are lactobacillus-specific cassettes out there; I didn't bring my lab notebook to Germany so I don't have the list with me, but I have a list of candidates. I plan to work with a supplier on the design of the eventual plasmid. We haven't decided whether to clone from an existing species ourselves, get someone else to do it, or have the gene synthesized from scratch, but I am leaning toward one of the latter two because I don't have the containment facilities to work with Pseudomonas or with Acidovorax avenae. (A. avenae is a plant pathogen, but it's a citrus pathogen and I live in California -- not inclined to take that risk.)
Where are you getting your primers?
When I do, it'll be from IDT. I don't think I'll get an ex-employee discount, but I love the company. :)
How will you choose your primers? Do you know how to design the construct and primers so that it will anneal successfully?
http://scitools.idtdna.com
Which restriction enzymes are you using?
Not at that stage yet. I plan to follow the lead of the BioBricks folks, though that will present its own interesting challenges, since BioBricks are designed for E. coli.
Are you using the shampoo/isopropyl alcohol method for DNA prep?
I've done it before, but the purity is kind of lousy. However, since I'm not at the point of cloning anything out of successfully transformed cells, that's another bridge to cross when I get to it.
no subject
What's your protocol?
For heat shock (which hasn't worked so far) I've tried a protocol from a patent that specifically focuses on lactic acid bacteria; it's a modification of an established protocol using PEG-8000, and I'll dig up the resource if you want to read it.
The heat shock protocol was pretty complicated, so recently I've been trying sonication, as described for E. coli here (http://nar.oxfordjournals.org/cgi/content/full/35/19/e129). No luck so far, though I'm going to do some more runs with better controls, and I'll try varying other aspects too -- so far I've only modified time, but I can also try temperature, CaCl2 concentration, adding some PEG, who knows; I need to do some more reviewing of the literature to get ideas, and have been keeping my eye out for a friendly microbiologist who works with Gram-positive bugs.
I would of course prefer to use electroporation, which was my old boss's suggestion, but electroporators are expensive. In addition to the wet-lab stuff I've been doing, I've also been working on a DIY electroporator; the logic side works, been having some problems getting the HV side to light up but that's what electrical-engineer friends are for. When I do it, I'll follow the protocol in "Optimization of technical conditions for the transformation of Lactobacillus acidophilus strains by electroporation", Kim, Han, Oh, You and Kim, J. Appl. Microbiology 2005.
Which plasmid/vector system?
At present, since I'm at the "get any old plasmid in there" stage, I have pGREEN plasmids from Carolina Biological.
Naturally, when we start trying to bang melamine deaminase in there, we'll need to build a custom plasmid for that. There are lactobacillus-specific cassettes out there; I didn't bring my lab notebook to Germany so I don't have the list with me, but I have a list of candidates. I plan to work with a supplier on the design of the eventual plasmid. We haven't decided whether to clone from an existing species ourselves, get someone else to do it, or have the gene synthesized from scratch, but I am leaning toward one of the latter two because I don't have the containment facilities to work with Pseudomonas or with Acidovorax avenae. (A. avenae is a plant pathogen, but it's a citrus pathogen and I live in California -- not inclined to take that risk.)
Where are you getting your primers?
When I do, it'll be from IDT. I don't think I'll get an ex-employee discount, but I love the company. :)
How will you choose your primers? Do you know how to design the construct and primers so that it will anneal successfully?
http://scitools.idtdna.com
Which restriction enzymes are you using?
Not at that stage yet. I plan to follow the lead of the BioBricks folks, though that will present its own interesting challenges, since BioBricks are designed for E. coli.
Are you using the shampoo/isopropyl alcohol method for DNA prep?
I've done it before, but the purity is kind of lousy. However, since I'm not at the point of cloning anything out of successfully transformed cells, that's another bridge to cross when I get to it.
(continued next comment)