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[Julia Doh]

I'm an undergrad working on a fimbrial protein mutagenesis project and I never received formal training in molecular biology techniques and my project doesn't have a supervisor so I'm getting pretty frustrated at not knowing how to troubleshoot any of my issues at work...

Basically I've been given a plasmid system where the target gene for mutagenesis has been inserted into a vector called Puc18-CM which is a chloramphenicol resistant variant of PUC19. The gene is on the lac promoter and is supposed to be inducible with IPTG. I've done all my mutagenesis and I've been trying to run experiments on them for a while now but I have an issue with overexpression (really long fimbriae that are way too big to be used in an atomic force microscopy experiment) even without induction so now my PI has asked me to verify my sequences to check if there is a defect in the promoter region.

The issue is however! Is that I don't have a very good map of my non-commercial vector Puc18-CM (it's just a hand-drawn picture and it just indicates the Plac comes before the lac gene). I was told the promoter is the same as the PUC19 promoter. I've been googling it but I've been seeing tons of different things and in whichever case at first glance many of them don't seem to match up to my sequences at all. It's supposedly -30 from the start of my gene but I've been having issues figuring out what I'm supposed to be checking. Normally to verify I translate my sequence to get an AA sequence using Expasy (so I can also see if my sequencing reaction ran backwards or forwards...) and then I do a protein BLAST and just compare it to literature sequence for FimA and then I can check for my mutations that way.

TL;DR: is there anyone who is familiar with PUC19 who can share with me the sequence and relative position of the lac promoter region? My only goal is the check that it's there and that it looks normal.

[bob1]

Here's the map and some information for pUC19  puc19_map.pdf   247.41K   161 downloads  pUC19_fasta.txt   3.59K   119 downloads