Hi,

I am not sure about your experimental design but you may consider the following.

One safe and effective way is to insert a plasmid containing a antibiotic resistance into your E. coli cells, which is quite easy. The process of inserting a plasmid DNA into a bacterial cell is called transformation. Upon successful transformation, the E. coli strain will be resistant to the specific antibiotic and you would be able to easily pick them using the antibiotic selection. Once it passes through the scavenger gut, you can easily look for e.coli strains resistant to the same antibiotic. If the E. coli has survived the journey through the scavenger gut it should show up in the faeces and  during  subsequent antibiotic screening. 

I hope it helps

Girish

Hello, 

I don´t know what is the goal of your experiment, but as Girishkumar Kaitholil ·said you can transfrom your E. coli with a plasmid, and even with a plasmid containing the GFP protein if you want to "see" bacteria. But if you just want to detect your E. coli in faeces maybe you can run a PCR or a Q-PCR to search and/or quantify your population. I don´t know if it is easy or not because you would need to find a sequence which was present solely in your strain. 

Hope this was usefull to you

As the others have said, we probably need to know more about the goal of the experiment--what exactly you are testing and what you will be comparing. Also, when you say "identify the marked cells", what type of equipment will you be able to use, microscope? flow cytometer? culturing methods? Do you need to use "your" E. coli strain or could would you be willing to purchase/obtain a strain that has the desired characteristics you are looking for, such as being able to pass through the gut and having a tag you can detect by either PCR/qPCR or by fluorescence.

I think introducing a plasmid DNA encoding GFP would be straight-forward (and ATCC has a strain with a GFP plasmid on it already for ~$300), but the E. coli may quickly lose the plasmid in the animal if it is not given enough ampicillin to maintain the plasmid. That would likely alter the gut flora and you might not get a good readout. Having a GFP or Enhanced GFP (EGFP) expressed from the bacterial chromosome instead might be a good way to go to maintain the gene. You could potentially make a fecal homogenate and put dilutions of it onto petri dishes with the appropriate growth medium and then visualize the colonies under a hand-held or other UV source. Your E. coli should be the only green-fluorescent ones. See the link for a paper and photo (Fig. 3a) that shows E. coli colony GFP expression either from the chromosome or from a plasmid (brighter, but may not be stable in vivo). Making dilutions and counting fluorescent colonies would allow you to enumerate the number of E. coli making it into the feces.

http://bioball.com/wp-content/uploads/technical_articles/Fluorescent%20reference%20strains%20of%20bacteria%20by%20chromosomal%20integration%20of%20a%20modified%20green%20fluorescent%20protein%20gene.pdf

The only way to mark your E. coli is to genetically modify it, as the other respondants have suggested. A plasmid expressing green fluorescent protein would seem to be ideal. However, you will need an antibiotic resistance gene as well, as the transformation process is quite inefficient in terms of the proportion of cells transformed. 

Your suggestion of labelling the E. coli with food coloring wouldn't work, because the E. coli cells will reproduce and multiply in the gut of the target organism. Any that does not multiply together with the cells will be diluted to undetectability. Thats why you need to use genes. 

If you do use a plasmid, make sure that it is entirely transfer deficient. Natural plasmids are easily exchanged between cells - often even between different species. That can result in your marker becoming separated from your marked cells. Synthetic plasmids, such as cloning vectors, are (usually) designed to be non-transferable in order to keep cloned DNA from spreading from laboratory strains. A GFP plasmid would probably conform to this specification, but do check.

Another approach would be to use genetic fingerprinting. Most bacterial strains can be distinguished by cutting their DNA with restriction enzymes. These enzymes cut the DNA at specific sequences and generate a pattern of fragments that can be separated by gel electrophoresis. The resulting pattern looks rather like a barcode and can be used in much the same way. The problem here is how to distinguish your E. coli from the animal's own natural E. coli flora. It's possible to use a brute force approach and fingerprint large numbers of E. coli colonies, but it's hard work.

Lastly, if you take the plasmid approach, be aware that natural strains of E. coli are much more difficult to transform than the laboratory strain, K12. K12, on the other hand, survives very poorly in the gut.  

Hi Christopher,

I'm hoping you may still be able to help us this.  Is it possible for you to contact me at ogada.darcyATperegrinefund.org?

Cheers, Darcy