4 replies to this topic

[Chris22]

Hi guys and girls

Posted here before and got great help so hope i will again!

Im looking to electrotransform a Lactobacillus with a dual plasmid system comprising of 2 vectors. (7000bp and 4000bp approx.)

It is a wild type lactobacillus isolated by myself.

1)   Glycine shocking o/n cells in 0.3M glycine MRS. Making competent cells by washing in ice cold SM liquid (sucrose and mgcl2), resuspending in the same.

2) Electroporation conditions: 1.5-2.0 kv, 25uf capacitance and 800 ohms resistance (due to high conductivity of SM liquid). 2mm gap cuvettes

3) Leaving for 3 hours without Ab selection in MRS (sucrose and mgcl2 enriched) broth.

4) Selecting on chloramphenicol and erythromycin MRS plates (varied concentrations a lot)

Ive managed to get each plasmid in seperately (but at extremely low efficiency.....like one clone!!!!), but cant a single clone with both!!!

I use the cells with one plasmid in already to make competent again.....and pulse with the other plasmid rather than both at the same time.


Im wondering if there is something wrong with my general technique, conditions or anything at all! If anyone has done this before or has any tips for pulsing etc i would much appreciate them!!!

Thanks in advance.

[Chris22]

As im not getting any replies, id settle for any general tips on making competent/transforming gram positive bacteria similiar to lactobacillus?? Im pretty much all out of ideas to make it work.

Thanks

[pDNA]

First of all i must confess i'm not that gram-positive specialist, but i'll try to help you anyway!

1) What origin of replication do your plasmids have? Do they have the same origin? ...for gram negative bacteria it is known that there are incompatibility groups of plasmids ...this means for example two plasmids from the same group can not coexist in one cell.

2) I know that some gram-positive like lactobacillus do have a S-layer, therefore it could be useful to digest with a low concentration of Proteinase K prior to making the cells competent (i'm sure you will find more detailed advice in the literature). Additionally, maybe it is possible to starve them before making competent to reduce the amount of exopolysaccharide they produce.

In general you can search for generation of sphero- and protoblasts ...i can remember that i read about an efficient protocol for lactobacillus using PEG-protoblasts.

Good luck!

Best regards,
p

[Chris22]

Hi pDNA

Thanks for replying.  

The origin of replication for each of the plasmids should not be an issue, they are part of an induction system and have been used extensively together before, as well as in other gram positives but not in lactobacillus.

I hadnt thought of using the PEG or the proteinase K method before, i assumed the glycine was enough to weaken the cell wall...maybe it is a case of the transformation method is fine but the cells are not being made competetent, or at very low efficiency.

I will try both this week and fingers crossed.

Thankyou for your input.

Chris22

[phage434]

With the mycoplasmas, the OD of the cell culture matters a great deal.  You might try spinning down some cultures that have much lower OD than you are accustomed to working with.  I would certainly start with the cells you have successfully transformed with one of the plasmids, since you are half way there.

I do think you want to know what your vector origin is, and what issues there are in multiple plasmids containing the same origin.  Is it a pWV01 origin, or other single stranded DNA intermediate vector?