Do you have an Aeromonas strain that's been reported to be transformed by this method as a positive control? That could help narrow down the issues to the strains you're working with instead of more upstream issues like someone giving you the wrong plasmid on accident or something.

Alexandra Johnson Unfortunately I dont have any positive strain. The plasmid can be transformed to DH5 alpha and give fluorescence.

Uthpala Chandrarathna how many transformed colonies have you screened for expression?

and during transformation try to give heat-shock for 90 seconds and then immediately transfer those comp cells to ice.

I have also tried to tag 1 of my fungal strain with GFP, after transformation primary culture were not showing any expression bt after secondary culture of the same transformants they were showing expression, that may be due to stress conditions they were in during transformation.

Ravi Shankar Gautam After transformation I immediately transferred them to ice. Next time i will increase the heat shock time. Many times i didn't even get colonies after transformation. And I used the florescence microscope to observe the GFP and but there was no GFP,. So did plasmid isolation for a few colonies and found no plasmid. So didn't do secondary culture.

I didnt know that GFP might express following cultures, thank you for sharing that information.

And I'm wondering why i can't get any colonies most of the times after transformation.

Uthpala Chandrarathna dont transfer to ice immediately after transformation they have to kept in 37 degree, transfer to ice after Heat shock only...

Best of luck...

Ravi Shankar Gautam I followed the steps you mentioned. still i didn't succeed however. Could you please tell me which type of plasmid you used for the GFP expression in your experiment

Uthpala Chandrarathna I have used pPdcEx PyrF RNAi Vector for GFP taging in Mucor.

Hi Uthpala,

Have you checked your plasmid for GFP expression after induction?. This plasmid contain Lac_promoter. So try after induction with IPTG. In the other hand, this plasmid may work well in E. coli but not in A. hydrophilia. Better you check it.

Thank you.

Amal Senevirathne PGFPuv has been used in some earlier studies to tag E. tarda and A. hydrophila. I found some references. I used IPTG but didnt get positive colonies.

Then check your plasmid in E.coli with IPTG induction. That should give you an idea about the level of expression. If that too does not give you a positive response, there should be a problem with the plasmid.

The RNA polymerase found in A. hydrophilia must be inefficient in interacting with lac_promoter of the PGFPuv plasmid. Normally they work best with E. coli RNA polymerases that is engineered in strains such as BL21 (DE3). Even you get colonies with some expression, that may not be sufficient for in vivo studies in A. hydrophilia.

Thank you.

Amal Senevirathne I used IPTG with E-coli and it worked. I checked the expression levels and pretty bright green fluorescence was developed with the time and after around 2 weeks no fluorescence detected even though the colonies had the plasmid.

Could you please suggest any alternative method to incorporate fluorescence tagged plasmid with A. hydrophila?

Mmmm, I think there must be stable covalent fluorescent tagging systems available. Or otherwise, if you can try little bit of cloning activity, you can try switching the promoter sequence to one of constitutively expressing promoter.