Single Molecule Microbiology

Congratulations to João Silva and everyone else in the lab who contributed to the first manuscript arising from his masters work. The manuscript, “Plasmids for independently tunable, low-noise expression of two genes,” is now available on bioRxiv. We characterized new plasmids that can be combined to tune the expression of two different genes in E. coli, and we discuss how these plasmids can be used to improve a variety of microbiology experiments. We also showed some data for single-molecule mRNA detection using the bacteriophage PP7 coat protein. The figure below shows how we can use this system to change the expression levels of GFP and mScarlet-I independently.

The manuscript was written using Manubot, a revolutionary tool for composing scientific manuscripts online.

New plasmids from the lab are now available at AddGene. First, a collection of plasmids for RNA imaging in E. coli using the coat protein from the bacteriophage PP7 fused to different fluorescent proteins. These are based on work from a few papers from Robert Singer’s lab, and make it possible to observe single RNAs containing tandem repeats of a specific sequence with either a cyan or red fluorescent protein. Second, plasmids from João Silva’s Masters project are available for low-noise gene expression. See more information on those in this Twitter thread:

Here's some new plasmids that use autoregulation and bicistronic expression to achieve tunable, low noise gene expression (rather than all-or-none expression that you get with many bacterial expression systems) - https://t.co/mX9i4yFDVM

— Zach Hensel (@alchemytoday) December 5, 2018

Congrats to João Silva submitted his thesis today on “Engineering low-noise gene expression systems for single-molecule experiments”. João developed new gene-expression plasmids that are compatible with ones that we previously reported. He showed that LacI/IPTG can work just as well as TetR/tetracycline, and that the systems can be moved to a compatible plasmid backbone with lower copy number. The plasmids are on their way to AddGene. The image shows how expression can be tuned to have low noise with only a few hundred molecules per cell in order to see spots that are labeled with a couple dozen molecules above background.

In the first selected talk for the group, Zach presented recent work by the Single Molecule Microbiology lab on new techniques for single mRNA imaging at the 84th Harden Conference on Single Molecule Bacteriology at Oxford University. His talk was titled “Improved tools for single-molecule mRNA and protein detection in living E. coli.”

Welcome to Sara Costa who recently joined the lab and presented her project today for the MolBioS PhD Program! Sara is co-supervised by Mariana Pinho. Her project is “Understanding the role of gene expression regulation in controlling cell division in Staphylococcus aureus.”