Hi all,
I want to express a ~100 bp single-stranded DNA sequences in E. coli. The project is to make a molecular beacon that has a ssDNA fragment making a hairpin that unfolds in response to a particular signal.
What's the best way to make ssDNA in E. coli? I know that M13 phage-based ssDNA is possible but are there any others methods?
Thanks
Mark
interesting question.
M13 indeed produces ssDNA molecules covered by capsid protein and slows down cell cycles to produce phage particles. some vectors have F1/mM13 origin to produce ssDNA using a helper phage M13KO7 . that was developed to get ssDNA from plasmids for sequencing and mutagenesis..
expression of ssRNA be easy , ss DNA hmmm
perhaps you can think about some plasmids wtih rolling cycle replication mechanisms put your fragment below ori and a kind of stop sequence, or add a specific site for ssDNA nuclease like Article XcmI as a universal restriction enzyme for single-stranded DNA
, or target this ssDNA with crisp/cas system...good luck
Hello,
such systems exist and can be used for what you want:
ref: Article DNA Enzyme Generated by a Novel Single-Stranded DNA Expressi...
In this paper they used moloney murine leukemia virus (MoMuLV) RT gene for reverse transcription and produced a DNA enzyme that targets an essential gene in E.coli. In your case you just have to change the sequence for the DNA enzyme to become that of your probe (the entire system is build to be contained on a single plasmid).
this has also been done with other system both in mammalian cells and yeast for example systems based on Retron-Ec67, a retroelement from E.coli. I have attached some additional references to help you decide what is best for your purpose:
https://www.future-science.com/doi/10.2144/03341dd04?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dwww.ncbi.nlm.nih.gov
Article Production of single-stranded DNA in mammalian cells by mean...
Best of luck!
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