Translating New Synthetic Biology Advances for Biosensing Into the Earth and Environmental Sciences (Valle et. al)

biosensors

genetically-engineered microbes/stand-alone biological components that sense and report on specific environmental conditions

benefits of using biosensors

they convert challenging and expensive signals into easily detectable outputs; report on micron-scale microbial experiences that are not available using common analytical approaches

metagenomics

who is there, biosensors can identify who is metabolically active

transcriptomics

what genes are expressed, biosensors can identify when and where are genes expressed

proteomics

what proteins/enzymes are present, biosensors can identify what enzymes are functional

metabolomics

what metabolites are present, biosensors can identify what metabolites are bioavailable

-omics vs. biosensors

whole community activities vs. specific microbial activities in time and space

what are the -omics

metagenomics, transcriptomics, proteomics, metabolomics

traditional use of biosensors in environmental research

detection of pollutants

what do biosensors consist of?

sensor, processing, and output module

sensor module

detects one or more environmental conditions as inputs

processing module

performs calculations using input signals

output module

produces detectable and quantifiable signal

DNA circuit diagrams

depict biosensors with information about genes in each module, regulation of gene expression, and module component interaction

shape of relationship between bionsensor output and input

sigmoidal

dynamic range of a sensor

difference between limit of detection and maximum input before saturation

limit of detection

threshold environmentla input needed for activation

how can biosensors address environmental challenges

report on complex microbial interactions, study spatial heterogeneity in situ, record hard-to-observe reactions

cell-to-cell signaling mediated by chemical signals

quorum sensing

quorum sensing

use of the accumulation of diffusible biomolecular signals to regulate commuinty behavior

N-acyl homoserine lactones (AHLs_

sginals used by marine bacteria to regulate enzymes that decompose POC

POC

particulate organic carbon

how do AHLs module nitrogen cycling

regulate denitrification and transfer of nitrogen-fixing machinery among bacteria

how are AHLs harnessed in engineered system

help control formation of biofilms and flocs for wastewater treatment

bioavailable fraction of signals

concentration that a microbe experiences

challenges when studying bioavailability with traditional methods

extremely low-level chemical concentrations

types of bias introduced from traditional chemical extraction methods

overestimation due to apolar signals, destruction of sample, no observation in situ

use of biosensors in soil

non-invasive measurements of AHL through indicator gases, soil controls on growth of nitrogen-fixing symbionts, exchange of symbiotic plasmids through AHl, plant-microbe communication

how can biosensors report on complex microbial interactions

how the environment influences cell-cell signaling, quantify how HGT varies with environment

what are examples of environmental conditions that can influence the frequency of microbe-microbe interactions

hydration, heterogeneity

drawbacks of traditional methods of studying conjugative HGT

cannot distinguish between new transconjugants and cells arising from VGT; cannot provide information on transfer frequencies

approaches for fluorescent reporters for HGT tracking

donor strain represses fluorescence until transferred

applications of fluorescent reporters for HGT tracking

how environmental parameters impact rate and range of conjugation

how do microbes use spatial gradients

control movement via chemotaxis, coexist as community, perform metabolic reactions

chemotaxis

movement of organism in response to chemical stimulus

impact of spatial heterogeneity on denitrification and N2O production

metal cofactors, nitrogen cycling depending on different oxygen requirements

methanogenesis and spatial variability

different environments (saturated soils, animal rumens, deep marine biosphere, engineered systems) and availability of electron donor sources

microbial AND gates

report on conditions controlling methanogenesis and denitrification: enzymes for carbon and nitrogen processing, proteins mediating metal uptake, bioavailability metal cofactors

cell-free biosensors

sense environmental input and produce a pigment or fluorescent output

cryptic reactions

fast chemical transformations in the environment that are hard to observe

why are iron and sulfur cryptic reactions important

related to decomposition of organic matter and production of greenhouse gases

sulfur cycle

controls ~29% of carbon minearlization in marine sediments

iron cycle

hard to track due to rapid biotic and abiotic reactions

how to leverage synthetic biology to study cryptic cycling

coding “spy” microbes to produce an easily measurable output when detecting a hard-to-observe metabolite; engineering microbes participating in cryptic reactions as biosensors, record information about production of a cryptic chemical before consumption

essential functions of biosensors

respond to environmental condition and translate response to output

intracellular sensors

protein or Rna switches engineered to sense environmental condition and a couple sensing to change in transcription

transcription factors

undergo conformational shift when analyte binds

riboswitches

RNA switches; use analyte binding to drive conformational change to affect gene expression

two component systems (TCSs)

extracellular sensors consisting of membrane-bound kinase that senses conditions and transmits info to intercellular response regular protein

kinase and gram-negative and gram-positive bacteria

unlike like gram-positive, gram-negative bacteria have kinase on the inner membrane, so analytes muss pass through pore protein before binding

visual reporters

fluorescent proetins, luminescent proteins, pigment-producing enzymes

drawbacks of visual reporters

hard to use in opaque materials

alternatives to visual reporters

ice nucleation protein, gas reporters, gas vesciles

ice nucleation protein (INP)

catalyzes ice formation from supercooled water; single channel; aerobic and aerobic sample conditions

gas reporters

gas chromatography mass spectometry is used to detect volatile gases

CH3X

CH3Cl, CH3Br, CH3I; used to report on conjugation in soil and activity of sensing module

C2H4

used to report number of microbial biosensors used to perform sensing; requires oxygen

gas vesicles

protein-structures filled with gas used by cyanobacteria to regulate buoyancy; used for contrast agent

application of logic gates

multiple inputs into a single output

biological memory

method by which cells can be programmed to record their past experiences by converting sensing information into heritable changes; permanent or rewritable

rewritable memory

recorded in a way that enables it to be reset; a pair of output states is used to indicate the most recent experience of a cell

permanent memory

written into the microbe’s DNA in a way that cannot be reversed; DNA recombinases that catalyze irreversible sequence inversinos

tuning input specificity

biosensors must respond specifically to signal of interest

improving tuning input specificity

random mutagenesis

tuning input sensitivity

biosensors must report on environmentally-relevant concentrations of the targeted analytes

improving input sensitivity

increasing or decreasing amount of sensor protein synthesized

cellular chassis

stripped down microbe intended to support engineered genes

challenges of introducing foreign DNA into undomesticated microbes

DNA transformation requires individual strian cultures

requirements for functioning of synthetic circuits

modules must be synthesized using regulatory elements for transcription (promoters) and translation (RBSs)

approaches to building fucntional genetic circuits

use of native promoters and RBSs, implementation of non-native regulatory elements that function independently from native system, use of regulatory elements that have similar funcitons across different organisms

identifying native machinery with a range of activities

single-cell transcriptomics

orthogonal biomolecular machinery

promoter and polymerase pairs that are functionally orthogonal to those in native organisms

broad-host range elements

regulatory elements that present similar activities across diverse organisms

examples of newly-programmable microbes

predatory soil and sediment bacteria, pollutant-degrading bacteria; gut bacteria; photosynthetic microbes; plant growth promoting rhizobacteria; bacteria associated with marine phytoplankton and animals; metal-reducing bacteria; bacteria from volcanic springs and hydrothermal vents

cell-free biosensors/transcription-translation reactions/ TX-TL reactions

contain lyophilized biomolecules that require hydration to use

cell-free biosensor design

cell extracts are prepared from lysed cells containing transcription and translational machinery; DNA program is added along with nutrients

field use of cell-free biosensors

field measurements of zinc and copper in municipal water, quorum sensing

requirements for biosensors to be useful for answering environmental questions

specific and sensitive sensing modules, production of measurable outputs, function of environmental study scales, use microbial strains of relevance, uitable for safe field applications

how can research expand inputs to biosensors

expanding the suite of sensing systems

how can research expand for outputs of biosensors

make them more environmentally-compatible

how can research be expanded for processing modules

recording complex information in situ

how can research be expanded for tuning

designing sensors that function across longer time scales

how can research be expanded by chassis

engineering more environmentally-relevant microbes

how can research be expanded for field deployment

engineering safe and reliable biosensors