[SCAN] Imaging single mRNAs and their encoded proteins in living Escherichia coli

Title:
Imaging single mRNAs and their encoded proteins in living Escherichia
coli

Abstract:
In the mid-2000s, transcription and translation were detected separately
at the single-molecule level in living Escherichia coli cells. However,
it has been challenging to combine these techniques in living bacteria.
A key limitation has been aggregation and apparent immortalization of
mRNA bound by fluorescent proteins. I report improved mRNA detection
methods using the bacteriophage PP7 coat protein to observe the
expression of tagged mRNAs at low expression levels. We've shown that
the appearance of single mRNAs can be correlated with bursts of protein
expression, and we're using 3-color smFISH to test the accuracy of
live-cell mRNA detection. The methods we will introduce can be applied
to address issues such as the extent of transcriptional bursting, the
transertion hypothesis, and fluorescent protein maturation rates. We're
also working to find out how the choice of fluorescent protein impacts
immortalization in PP7 coat protein fusions. Lessons learned in PP7
likely apply to MS2 and similar systems based on dimeric RNA-binding
proteins. RNA imaging methods that work in bacteria are likely to be
useful in other organisms that pose fewer technical challenges (e.g.
larger cell volumes and longer mRNA lifetimes). Additionally, I will
talk briefly about other projects in the lab and present some
instruments and techniques we’ve recently introduced in our lab that
might be of interest to other labs at ITQB — 3d microscopy with
adaptive optics, 3d printing of optomechanics components, and using deep
learning for image segmentation.