King Jonathan

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King
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Jonathan
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  • Article
    High throughput instrument to screen fluorescent proteins under two-photon excitation
    (Optical Society of America, 2020-12-01) Molina, Rosana S. ; King, Jonathan ; Franklin, Jacob ; Clack, Nathan ; McRaven, Christopher ; Goncharov, Vasily ; Flickinger, Daniel ; Svoboda, Karel ; Drobizhev, Mikhail ; Hughes, Thomas E.
    Two-photon microscopy together with fluorescent proteins and fluorescent protein-based biosensors are commonly used tools in neuroscience. To enhance their experimental scope, it is important to optimize fluorescent proteins for two-photon excitation. Directed evolution of fluorescent proteins under one-photon excitation is common, but many one-photon properties do not correlate with two-photon properties. A simple system for expressing fluorescent protein mutants is E. coli colonies on an agar plate. The small focal volume of two-photon excitation makes creating a high throughput screen in this system a challenge for a conventional point-scanning approach. We present an instrument and accompanying software that solves this challenge by selectively scanning each colony based on a colony map captured under one-photon excitation. This instrument, called the GIZMO, can measure the two-photon excited fluorescence of 10,000 E. coli colonies in 7 hours. We show that the GIZMO can be used to evolve a fluorescent protein under two-photon excitation.
  • Article
    Protein folding failure sets high-temperature limit on growth of phage P22 in Salmonella enterica serovar Typhimurium
    (American Society for Microbiology, 2004-08) Pope, Welkin H. ; Haase-Pettingell, Cameron ; King, Jonathan
    The high-temperature limit for growth of microorganisms differs greatly depending on their species and habitat. The importance of an organism's ability to manage thermal stress is reflected in the ubiquitous distribution of the heat shock chaperones. Although many chaperones function to reduce protein folding defects, it has been difficult to identify the specific protein folding pathways that set the high-temperature limit of growth for a given microorganism. We have investigated this for a simple system, phage P22 infection of Salmonella enterica serovar Typhimurium. Production of infectious particles exhibited a broad maximum of 150 phage per cell when host cells were grown at between 30 and 39°C in minimal medium. Production of infectious phage declined sharply in the range of 40 to 41°C, and at 42°C, production had fallen to less than 1% of the maximum rate. The host cells maintained optimal division rates at these temperatures. The decrease in phage infectivity was steeper than the loss of physical particles, suggesting that noninfectious particles were formed at higher temperatures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a decrease in the tailspike adhesins assembled on phage particles purified from cultures incubated at higher temperatures. The infectivity of these particles was restored by in vitro incubation with soluble tailspike trimers. Examination of tailspike folding and assembly in lysates of phage-infected cells confirmed that the fraction of polypeptide chains able to reach the native state in vivo decreased with increasing temperature, indicating a thermal folding defect rather than a particle assembly defect. Thus, we believe that the folding pathway of the tailspike adhesin sets the high-temperature limit for P22 formation in Salmonella serovar Typhimurium.