tihong10

Hello,

I was wondering if someone with more knowledge than me in the area of measuring fluorescent intensity of fluorescent proteins can shed some light on this new topic. I am a newbie in this field so I don't know precise terminology but I will try to make sense:

I have transformed bacteria containing a plasmid with a promoter-RFP (red fluorescent protein) fusion that I am using as a reporter of gene activity. The RFP is dsRed from dsRed-Express2 plasmid, and it has an excitation maximum of 554nm and an emission maximum of 591nm. My question is do I need to set the excitation and emission values exactly on the machine (a PerkinElmer Victor x5 multilabel plate reader)? Is there one that I must set exactly right?

We currently don't have filters that exactly match those numbers so I am running my experiments without them but I am not getting good results. I wanted to know if the settings were the problem or some other aspect of my experiment may be it.

Thank you,
Ted

Hi, I electroporated P. aeruginosa 14 with a plasmid and when I tried to view the plasmid extraction in a gel it came out as a smear. Is there some big issue I am not aware of when plasmid extracting from PA? Is my plasmid being degraded somehow?

I wasn't sure how much detail to put but if more is needed I will provide it.

Thanks!

Hi everyone,

I want to insert a reporter (red fluorescent protein) into the genome of P. aeruginosa to study the activity of a certain promoter. I was wondering if there was a method of making a specific insertion into the genomic DNA of a bacterium; if so, please let me know about it. Any suggestions or articles welcome.

Thank You!

Hi everyone,

I want to insert a reporter (red fluorescent protein) into the genome of P. aeruginosa to study the activity of a certain promoter. I was wondering if there was a method of making a specific insertion into the genomic DNA of a bacterium; if so, please let me know about it. Any suggestions or articles welcome.

Thank You!

Hello All,

I was performing a tranformation of DH5a with what I thought was a legitimate recombinant plasmid. The vector* i.e. plasmid without the insert has an AMP marker and a lacZ-red fluorescent protein fusion with a MCS in between the lacZ and RFP. After adding my ligation mixture with the chemically competent DH5a I plated on carbenicillin plates. Upon confirmation, I observed no growth on the control (non-transformed DH5a) plates and found two types of growth on the experimental plates. One colony type was cream colored and the other was red. I initially thought the cream colored colonies contained the recombinant plasmid because I was under the impression that an insertion in the multiple cloning site will severe the fusion between the lac promoter and RFP. However, I found out later that my primers were constructed incorrectly so that one of the enzyme sites was unrecognizable by the restriction enzyme.

Thus I am left wondering why clones grew at all on the antibiotic plates. If my insert and plasmid were double digested but didn't correctly cut one of the two restriction sites then how did colonies grow on the plate? Also, how does one explain the appearance of cream and red color colonies?

Also, am I right to assume that an insertion into the multiple cloning site of the original vector will result in no RFP production when the lac promoter is active?

If anyone can provide any insight into these questions I'd highly appreciate it.

THanks!




*Map of Plasmid Vector
http://www.snapgene....DsRed-Express2/