Question 1:

Do mean that the plasmid carrying the actual lamda red system or do mean doing lamda red on a plasmid that has a cole1 origin?

A1: if you mean the plasmid that carries the lamda red system, low copy number plasmids are used because the expression of lamda red is toxic hence low copy plasmids are more suited for this. Also the plasmids used in lamda red for this purpose carry a modified rep protein that is temp sensitive hence the plasmid can be cured quickly. Plasmid pKD46 or pSIM series of plasmids are recommended.

A2:if you mean you want to carry out lamda on a plasmid that has a cole1 origin then that is fine as along as you introduce a marker that allows the isolation of the correct recombined plasmid.

Q2- Chemical transformation vs electroporation:

A1: lamda red requires a high intracellular amount of DNA, which achieved much more efficiently using electroporation. However, it is possible to achieve this using chemical transformation, I have only found one combination that works, in this case you should be using a very high amount of DNA to transform (microgram range) combined with high expression of lamda red using plasmid pSIM6.

Q3- Maintenance of RED proteins:

Feel free to make competent cells that you store in the fridge after RED induction. Once the cells are frozen at -80C the proteins will remain stable and should be still available to carry out their function for the duration of the transformation protocol (which is very short if using electroporation).

Link to pSIM plasmid series:

https://redrecombineering.ncifcrf.gov/strains--plasmids.html

Hi Hanna,

Thank you so much for your answers, they are really helpful.

I am carrying out lambda on a plasmid that has colE1 origin, so around 20 copies per cell. I am introducing a marker as an intermediate sequence that will contain tetracycline resistance and streptomycin sensitivity for positive and negative selection of my intermediate clones, respectively. However, I was thinking that maybe not all of the 20 plasmids within the selected cell will be recombineered, giving me a heterogenous population of recombineered+/- plasmids that can coexist in the same cell. If this happens, cells that are tetracycline resistance could still carry the original plasmid, right? Is this common?

I was also thinking of using E. coli strains that are already engineered to express the lambda system if induced, thus avoiding the extra transformation step of the Red/ET plasmid. Is this a better approach? do you have any recommended strains that will be efficient at recombineering a plasmid with 20 copies per cell, as most of the strains I have seen are suited for BACs (1-2 copies per cell)

Thank you so much again!

It is almost definite that you will have a mixed population. you will have to miniprep after lamda red and retransform. Your prep will contain both plasmid however if you select tet, the chances of a double transformation is low and you can confirm by double checking that the original resistance is not there anymore by growing the cells with that antibiotic. as for a strain for this you can check the link I sent, one of the options there is a strain that has lamda red in a chromosomal location.