Green Fluorescent Protein

[biomat]

Hi,

 

I know GFP plasmid can be easily introduced and expressed in E. coli but I am wondering if the same plasmid that works in E. coli will work in other species?

 

I would like to express GFP in Staph. epi. but I am no geneticist and I do not know if the plasmid has to be specifically tailored for the species. Any help would be appreciated.

 

My purpose is to observe bacterial attachment in situ by epifluorescent microscopy.

 

Thanks

biomatt

[claytonm]

Hi' date='

 

I know GFP plasmid can be easily introduced and expressed in E. coli but I am wondering if the same plasmid that works in E. coli will work in other species?

 

I would like to express GFP in Staph. epi. but I am no geneticist and I do not know if the plasmid has to be specifically tailored for the species. Any help would be appreciated.

 

My purpose is to observe bacterial attachment in situ by epifluorescent microscopy.

 

Thanks

biomatt[/quote']

 

Biomatt,

 

I am no expert on this but I have created a GFP mutant in Hylicobacter pylori for flagellar biogenesis studies, so I have somewhat of an idea. The reason GFP in a standard plasmid will work in E. coli is because the plasmid the GFP gene is located in contains an origin of replication (commonly referred to as ori , it will usually have a number designation that refers a research to its exact sequence since there is more than one origin of replication recognized by E. coli) that is recognized by the polymerase in E. coli and allow it to replicate i.e. the plasmid is passed to the progeny and not lost during replication. If you attempted to transform this same plasmid into Staph. epi., it will be lost after the first generation of cells since it can't replicate. To get around this you have to do what it referred to as homologous recombination. This basically means that the GFP needs to be cloned between a copy of gene found in the genome of Staph. epi. This Staph gene could be one used for attachment or creation of a biofilm. You will have to look at the genome and determine what genes are involved in attachment since I am not familiar with them. To do this you will have to amplify the GFP gene and Staph gene of interest through a mulit step SOE PCR. You will have to create 5' and 3' overhangs in the Staph gene and the GFP gene that allow them to be PCR amplified as one big fragment together. Also, you will have to attach some kind of phenotypic marker gene (usually antibiotic resistance) to the target gene that is not found in the vector. Then, ligate that fragment into a vector to create a plasmid that has some other phenotypic marker (could be antibiotic resistance or Lactose metabolism i.e. LacZ but needs to be different than the phenotypic gene attached to your target gene). You will then have to transform this new plasmid back into E. coli to screen for colonies that have taken up the correct plasmid ( when you ligate your fragment with the vector you can get lots of different plasmids) . The next step is very tricky stuff because you have to get the target gene out of the plasmid before putting it into Staph or you will be knocking out your gene of interest. This can be done by PCR amplifying the target gene (Staph gene + GFP + phenotypic marker gene), purifying it and then transform it into Staphylococcus epidermidis and screen it on selective media to determine which colonies have actually taken up the naked DNA. Colonies of Staph that have the correct phenotype should have homologously recombined the gene of interest that has the GFP and phenotypic marker, into the genome of Staph. Once it is in the Genome it can replicate like the rest of the genomic DNA. You can confirm this buy preparing some Genomic DNA from mutant Staph and then doing a PCR using a primer upstream of the target gene and a primer down stream of or target gene, as long as you know the expected amplicon size. Once you have confirmed that you have the correct mutant all you have to do is grow it up, properly prepare it for some microscopy and take some pretty pictures of your GFP. I consider this a very complicated thing to do and is very time consuming. All that being said, it is possible to do and scientists have done it over and over again. I will try to dig up some papers that have the exact methods involved in them as I'm sure I made some mistakes along the way (it's late and I wrote way too much). I hope this helps you a little bit.

 

-Clayton

 

P.S. What I described is used more for attaching GFP to a particular gene of interest to see where exactly the protein is expressed. I guess that it is completely possible to create a plasmid that contains an ori recognized by Staph that is can use to self replicate. This is only useful if you just want to see your cells fluoresce. Then all you would have to do is clone your GFP gene into the plasmid, throw it at Staph and away you go. I will have to look into this tomorrow.

[biomat]

Thanks for the informational reply. All of those techniques are familiar to me, it has just been a while since I have done any molecular biology.

 

Your last paragraph is correct, I am just looking to express GFP in Staph epi for in-situ viewing purposes. I am not really doing work on adhesive/biofilm proteins, my main objective is to test anti-fouling biomaterial surfaces. I put my surface in a flow cell and pump bacteria through it while recording with an epifluorescence microscope/imager. I want to make a time-lapse video so I can actually show the cells attaching/detaching under different flow rates.

 

Right now I am using a nucleic acid stain on the live cells in PBS but I would much rather be able to use GFP transfected cells. I can actually do this experiment with E. coli as they are somewhat adhesive but my Staph. epi. strain is a nice clinical isolate so I would love to be able to use that.

 

I have been looking into this for a couple weeks without much success. I will have to look into an origin of replication for staph epi. Thanks for the help and feel free to comment further.

[zyncod]

You know, if you're just trying to detect exogenous cells, a dye would probably be more appropriate than a transgene. We use use CFSE and CMTMR for labeling cells for subQ injection into mice. The false positive rate by flow cytometry for these dyes is 0-5/300,000 cells.

 

P.s.- For these amine-reactive dyes, you don't need to permeabilize the cells the way you might need to for a nucleic acid stain. Really, the stains are incredibly effective at very low concentrations as they are membrane-permeable and bind permanently to intracellular proteins.

[biomat]

Interesting. I will have to look into those dyes. Right now I am using Syto 9 (molecular probes) which is a bright nucleic acid stain that stains live and dead cells without need for permeabilization. I am just concerned about possible negitave affects on protein expression and cell attachment behavior.

 

Maybe I'm being overly cautious but this work is part of a larger project which will probably be published in a highly respected journal so I need to make sure I cover all bases. Thanks for the help.