4 replies to this topic

[kaylosin]

Hi,
I am new to the world of H. influenzae and interested in generating transposon library. I have several options like doing invitro transposon mutagenesis using EZ-tn5 system and recently I saw in vivo mutagenesis approach based on tn10. I was wondering if somebody already tried to contruct a transposon library using the vitro approach and can suggest me the right kit. Also I am having severe troubles in transforming Haemophilus. I am working with Haemophilus aegyptius.

[phage434]

I have no experience with Haemophilus, but have had experience with the Tn5 transposome system from Epicentre.  I think you'd find it easy to use, although the transposase protein is pricey.  Are you making a sequencing library of DNA fragments, or a library of transposon knockouts?

[kaylosin]

Hi,
Iam trying to make a knock out library. We have a phenotype and need the gene or protein reponsible for that.

phage434, on May 26 2009, 01:39 PM, said:

I have no experience with Haemophilus, but have had experience with the Tn5 transposome system from Epicentre.  I think you'd find it easy to use, although the transposase protein is pricey.  Are you making a sequencing library of DNA fragments, or a library of transposon knockouts?

[klinmed]

kaylosin, on May 26 2009, 03:06 PM, said:

Hi,
I am new to the world of H. influenzae and interested in generating transposon library. I have several options like doing invitro transposon mutagenesis using EZ-tn5 system and recently I saw in vivo mutagenesis approach based on tn10. I was wondering if somebody already tried to contruct a transposon library using the vitro approach and can suggest me the right kit. Also I am having severe troubles in transforming Haemophilus. I am working with Haemophilus aegyptius.

Haemophilus sp. can take up DNA naturally. The level of competency depends on growth condition (usually a shift of log phase cell to a medium that cannot support growth.  However, uptake of DNA is usually dependent on the presence of a common 11 bp sequence.

Electroporation has been shown to be very effective for transformation of hetero (no 11 bp sequence) plasmid DNA in H. influenzae. See:

Nucleic Acids Research, 1991, Vol. 19, No. 13 3625-3628

Hope this helps.

[kaylosin]

Hi,
Thanks for the info. But I am aware of this . I have been trying to transform H. influenzae using natural transformation procedure but I still dont get any transformants. I was trying to do an invivo transposition using a vector which has the 11 basepair USS sequence and transposon and transposase on it. Since the transforamtion itself doesnt work I cant reach to the point of transposition. But I will try electroporation!

klinmed, on May 28 2009, 02:38 AM, said:

kaylosin, on May 26 2009, 03:06 PM, said:

Hi,
I am new to the world of H. influenzae and interested in generating transposon library. I have several options like doing invitro transposon mutagenesis using EZ-tn5 system and recently I saw in vivo mutagenesis approach based on tn10. I was wondering if somebody already tried to contruct a transposon library using the vitro approach and can suggest me the right kit. Also I am having severe troubles in transforming Haemophilus. I am working with Haemophilus aegyptius.

Haemophilus sp. can take up DNA naturally. The level of competency depends on growth condition (usually a shift of log phase cell to a medium that cannot support growth.  However, uptake of DNA is usually dependent on the presence of a common 11 bp sequence.

Electroporation has been shown to be very effective for transformation of hetero (no 11 bp sequence) plasmid DNA in H. influenzae. See:

Nucleic Acids Research, 1991, Vol. 19, No. 13 3625-3628

Hope this helps.