10 replies to this topic

[joy123]

I want to do gene knockout in a bacteria. I tried several vectors, but none was able to be transformed into the bacteria.

Is it common that foreign vectors don't survive in wild type bacteria? Should I check which vectors can survive in the bacteria firstly before I start? Unfortunitely, my bacteria is seldom studied and it is hard to find references.

I am a new starter in this field and hope to get some help. Thanks for reading my poster, and look forward for any of your suggestions!

[pDNA]

are there any publications available on releated species? ...you need a vector that has an origin of replication that can be recognized by your bacteria (this means the promoters should work there). Maybe you find plasmids that have been already characterized and known for replicating in a lot of gram positive or negative bacteria.

Another problem could be interference with methylation patterns ...some bugs do not like the methylation pattern E. coli produces ...therefore plasmids get degraded upon transformation ...or restriction systems that degrade the plasmid you are transforming.

You see several possible reasons why its hard to transform wt bacteria ...i would do a in depth literature research before starting wet lab work!
What bug are you working with?

Regards,
p

[phage434]

As the saying goes, six months in the laboratory can save you an entire afternoon at the library.

I'd recommend looking at broad host range plasmids.  There are several out there.  Some notable ones are pAMbeta1 and pWV01.  You can check pubmed for articles on these.  (The first often has a greek beta, the second is ambiguous about whether the zero is a zero or an O).

[pDNA]

great saying

RSF1010 is also a prominent one if you are working on gram negatives!

Regards,
p

[joy123]

I like that saying:) I heared that someon spent half a year trying to do transformation. They have tried almost every possible conditions, but failed.

Thanks for your suggestions. I am working on reading, as I am new in this field.

Would you please give me some suggestions about what aspects about the bacteria should I read about? I am not very clear what I should regard before I design the experiment. Thanks a lot!

[joy123]

Hi, Thanks very much for your response.

What I am working on is a rothia species. They are seldomly studied. So I don't have much reference to go. One family member is genotyped. Still I don't know what aspects I should study about.

Besides, I see some people use cell mating. Is it easiler than plasmid/ linear DNA transformation?

And, how many ways are there to do gene deletion in bacteria? Up to now I have read about recombineering, in frame gene deletion, Targetron kit in sigma. I hope to get a big picture, and then choose a suitable one for my bacteria.

Thanks very much! Look forward for responses.

pDNA, on 10 March 2012 - 12:41 AM, said:

are there any publications available on releated species? ...you need a vector that has an origin of replication that can be recognized by your bacteria (this means the promoters should work there). Maybe you find plasmids that have been already characterized and known for replicating in a lot of gram positive or negative bacteria.

Another problem could be interference with methylation patterns ...some bugs do not like the methylation pattern E. coli produces ...therefore plasmids get degraded upon transformation ...or restriction systems that degrade the plasmid you are transforming.

You see several possible reasons why its hard to transform wt bacteria ...i would do a in depth literature research before starting wet lab work!
What bug are you working with?

Regards,
p

[pDNA]

if no literature is available on Rothia sp. i would go for related species, like Micrococcus (or whatever is releated ...you know better for sure!) ...or at least look for all transformation protocols for gram positive bacteria (should be not that many) ...and than consider what you know about your bug and than take the protocol for the most similar bug (outer membran composition, s-layer, whatever) ...and start with that and if necessary modify it step be step ...by adding different treatments and conditions ...but there is no generic approach on this ...its more or less trial and error based (this is the reason why we have seen so few protocols established for less studied organism).

Anyway, good luck!!!

Best regards,
p

[phage434]

I would start with electroporation as a baseline.  Wash the cells with water (ideal) or an iso-osmotic solution of a non-ionic compound such as sucrose or sorbitol.  You can check CFUs after washing to make sure you haven't killed the cells.  Cell growth stage can be a critical variable.  Easiest to try for knockout would be a knock-in assay.  Make a linear DNA construct containing an antibiotic selection cassette and 500-1000 bp region of homology to the desired insert location on each side of the cassette.  Purify the DNA, put lots of it in with your cells, and electroporate.  Culture without selection for 1-2 doubling times, then plate on selective medium.  You can check CFUs post-electroporation to test whether you are trashing the cells.  Life will be easier for you if you can freeze the cells after washing.  Try storing the cells at -80 in a 10% glycerol solution.

[phage434]

Also, you probably should characterize the restriction enzymes present in your organism.  Genome analysis may tell you something, but experimenting with cell lysates is the sure way to tell.  It would be good to either eliminate restriction sites, or to methylate your DNA to protect it.

[joy123]

Thanks a lot!

I am thinking about transferring linear DNA into my bacteria, instead of vectors. I have several questions regarding to this technique:

1. what is this technique called? I hope to search more about it.
2. is linear DNA easier than vectors? Does it also has the problem of "not matching the bacteria" and thus being degraded?
3. I have read a protocol constructing linear DNA and transform to another bacteria. And then they did cell mating to their interest bacteria. I want to know if mating is easiler than linear DNA and vector transformation?

Thanks very much for your time!

phage434, on 14 March 2012 - 09:13 AM, said:

I would start with electroporation as a baseline.  Wash the cells with water (ideal) or an iso-osmotic solution of a non-ionic compound such as sucrose or sorbitol.  You can check CFUs after washing to make sure you haven't killed the cells.  Cell growth stage can be a critical variable.  Easiest to try for knockout would be a knock-in assay.  Make a linear DNA construct containing an antibiotic selection cassette and 500-1000 bp region of homology to the desired insert location on each side of the cassette.  Purify the DNA, put lots of it in with your cells, and electroporate.  Culture without selection for 1-2 doubling times, then plate on selective medium.  You can check CFUs post-electroporation to test whether you are trashing the cells.  Life will be easier for you if you can freeze the cells after washing.  Try storing the cells at -80 in a 10% glycerol solution.

[joy123]

Would you please let me know how to do it? Or how should I search for the protocol or books?

Thanks a lot!

phage434, on 14 March 2012 - 10:14 AM, said:

Also, you probably should characterize the restriction enzymes present in your organism.  Genome analysis may tell you something, but experimenting with cell lysates is the sure way to tell.  It would be good to either eliminate restriction sites, or to methylate your DNA to protect it.