7 replies to this topic

[garciapp]

I need to put a stable vector with a single EcoRI site into Salmonella typhimurium, but it cannot be pUC/ColE1 based, because I am already maintaining a ColE1 plasmid in this strain, so I need a compatible vector. Does anyone have any suggestions for a good one to use? Preferably under 10Kb if possible.

Thank you,
Preston

[fishdoc]

garciapp, on Jul 27 2009, 02:27 PM, said:

I need to put a stable vector with a single EcoRI site into Salmonella typhimurium, but it cannot be pUC/ColE1 based, because I am already maintaining a ColE1 plasmid in this strain, so I need a compatible vector. Does anyone have any suggestions for a good one to use? Preferably under 10Kb if possible.

Thank you,
Preston

pBBR1

http://www.ncbi.nlm..../pubmed/8529885

[garciapp]

fishdoc, on Jul 27 2009, 03:56 PM, said:

garciapp, on Jul 27 2009, 02:27 PM, said:

I need to put a stable vector with a single EcoRI site into Salmonella typhimurium, but it cannot be pUC/ColE1 based, because I am already maintaining a ColE1 plasmid in this strain, so I need a compatible vector. Does anyone have any suggestions for a good one to use? Preferably under 10Kb if possible.

Thank you,
Preston

pBBR1

http://www.ncbi.nlm..../pubmed/8529885

Thanks, I was looking at that paper before, good to get confirmation. I'll see about getting one of those. Appreciate your help.

[fishdoc]

garciapp, on Jul 28 2009, 08:37 AM, said:

fishdoc, on Jul 27 2009, 03:56 PM, said:

garciapp, on Jul 27 2009, 02:27 PM, said:

I need to put a stable vector with a single EcoRI site into Salmonella typhimurium, but it cannot be pUC/ColE1 based, because I am already maintaining a ColE1 plasmid in this strain, so I need a compatible vector. Does anyone have any suggestions for a good one to use? Preferably under 10Kb if possible.

Thank you,
Preston

pBBR1

http://www.ncbi.nlm..../pubmed/8529885

Thanks, I was looking at that paper before, good to get confirmation. I'll see about getting one of those. Appreciate your help.

I've used it in a Gram (-) enteric as a complementation plasmid for a mutant, and it worked well. Am trying it now as a vector to carry a FLAG-tagged gene. We use E. coli CC118 lambda pir to carry it, but it is not lambda pir-dependent, so I'm not sure why that strain was used. I will be trying today to see if XL1-Blue MRF' will carry it or not.

It's got the MCS of pBluescript in it, so it's very flexible for cloning.

We conjugate it to our bacterium using SM10 lambda pir, but again, I'm not sure why that is... perhaps that strain is the only one we have that has the transfer genes... never really thought about it before until now.

[fishdoc]

fishdoc, on Jul 28 2009, 09:07 AM, said:

I've used it in a Gram (-) enteric as a complementation plasmid for a mutant, and it worked well. Am trying it now as a vector to carry a FLAG-tagged gene. We use E. coli CC118 lambda pir to carry it, but it is not lambda pir-dependent, so I'm not sure why that strain was used. I will be trying today to see if XL1-Blue MRF' will carry it or not.

Just looked at the plates, and it replicates fine in XL1-Blue MRF', and blue/white or black/white screening can be used.

[garciapp]

fishdoc, on Jul 29 2009, 09:25 AM, said:

fishdoc, on Jul 28 2009, 09:07 AM, said:

I've used it in a Gram (-) enteric as a complementation plasmid for a mutant, and it worked well. Am trying it now as a vector to carry a FLAG-tagged gene. We use E. coli CC118 lambda pir to carry it, but it is not lambda pir-dependent, so I'm not sure why that strain was used. I will be trying today to see if XL1-Blue MRF' will carry it or not.

Just looked at the plates, and it replicates fine in XL1-Blue MRF', and blue/white or black/white screening can be used.

Thanks for the updated info. I've put in a request to have the vectors sent to me, so hopefully I will be testing them out myself soon.

[garciapp]

I have all six of the pBBR1MCS plasmids, but I cannot get anything to go into them for some reason. Four of them with EcoRI sites and 2 with SmaI. I cannot figure out why such easy cloning has been so difficult. Did you have any trouble cloning anything into these vectors?

[fishdoc]

garciapp, on Oct 1 2009, 08:06 AM, said:

I have all six of the pBBR1MCS plasmids, but I cannot get anything to go into them for some reason. Four of them with EcoRI sites and 2 with SmaI. I cannot figure out why such easy cloning has been so difficult. Did you have any trouble cloning anything into these vectors?

No, I've never had much trouble. Can't recall ever using EcoRI or SmaI, but I've used KpnI, SacI, XbaI, and maybe more. What E. coli strain are you putting them into (I've gotten them into XL1 Blue MRF', CC118 lambda pir, and SM10 lambda pir).