Difference between revisions of "Proof of concept continuous event logging in living cells"

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{{Paper
 
{{Paper
 
|Title=Proof of concept continuous event logging in living cells
 
|Title=Proof of concept continuous event logging in living cells
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|Authors=Andrey Shur,  Richard M Murray
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|Authors=Andrey Shur,  Richard M. Murray
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|Source=2018 Synthetic Biology: Engineering, Evolution and Design (SEED) Conference
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|Source=2019 Synthetic Biology: Engineering, Evolution and Design (SEED) Conference
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|Abstract=Biological records are omnipresent in paleontology, history, and climate science. Tree rings and ice cores provide evidence of environmental conditions that have been recorded in the composition of materials that are deposited over time, carrying with them a record of events that have influenced their existence before being buried underneath ice or inside the trunk of a tree. We constructed a proof of concept synthetic circuit that can be used to create a similar chronological record of events in the DNA of a living E. coli. In our system, phage-based serine integrases are employed to sequentially integrate pieces of DNA corresponding to which stimulus is being detected. We show that placing attB and attP sites close together on a piece of DNA prevents intramolecular reactions, and enables repeated integration events to expand a genetic locus proportionally to integrase induction and abundance of plasmid DNA. We also show that dCas9 binding can prevent integrase from reacting with an attachment site, and in so doing we can control which piece of DNA is integrated by the induction of different guide RNAs. These results represent significant steps towards an event logger that is capable of recording the ordering and magnitude of any number of molecular events. Such a system may be useful in studying complex biological phenomena such as biofilm formation, quorum sensing, or signaling in the gut.
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|Abstract=Cells must detect and respond to molecular events such as the presence or absence of specific small molecules. To accomplish this, cells have evolved methods to measure the presence and concentration of these small molecules in their environment and enact changes in gene expression or behavior. However, cells don’t usually change their DNA in response to outside stimuli. In this work, we have engineered a genetic circuit that can enact specific and controlled genetic changes in response to small molecule stimuli. Known DNA sequences can be repeatedly integrated in a genomic array such that their identity and order encodes information about past small molecule concentrations that the cell has experienced. To accomplish this, we use catalytically inactive CRISPR-Cas9 (dCas9) to bind to and block attachment sites for the integrase Bxb1. Therefore, through the co-expression of dCas9 and guide RNA, Bxb1 can be directed to integrate one of two engineered plasmids, which correspond to two orthogonal small molecule inducers that can be recorded with this system. We identified the optimal location of guide RNA binding to the Bxb1 attP integrase attachment site, and characterized the detection limits of the system by measuring the minimal small molecule concentration and shortest induction time necessary to produce measurable differences in array composition as read out by Oxford Nanopore sequencing technology.
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|URL=https://www.biorxiv.org/content/early/2018/03/08/225151
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|URL=https://www.biorxiv.org/content/10.1101/225151v3
 
|Type=Conference paper
 
|Type=Conference paper
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|ID=2018a
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|ID=2019b
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|Tag=sm18-seed
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|Tag=SM19-seed
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|Funding=DARPA BioCon
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|Funding=ICB Network19
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|DOI=10.1101/225151
 
}}
 
}}

Latest revision as of 23:49, 9 June 2019

Title Proof of concept continuous event logging in living cells
Authors Andrey Shur, Richard M. Murray
Source 2019 Synthetic Biology: Engineering, Evolution and Design (SEED) Conference
Abstract Cells must detect and respond to molecular events such as the presence or absence of specific small molecules. To accomplish this, cells have evolved methods to measure the presence and concentration of these small molecules in their environment and enact changes in gene expression or behavior. However, cells don’t usually change their DNA in response to outside stimuli. In this work, we have engineered a genetic circuit that can enact specific and controlled genetic changes in response to small molecule stimuli. Known DNA sequences can be repeatedly integrated in a genomic array such that their identity and order encodes information about past small molecule concentrations that the cell has experienced. To accomplish this, we use catalytically inactive CRISPR-Cas9 (dCas9) to bind to and block attachment sites for the integrase Bxb1. Therefore, through the co-expression of dCas9 and guide RNA, Bxb1 can be directed to integrate one of two engineered plasmids, which correspond to two orthogonal small molecule inducers that can be recorded with this system. We identified the optimal location of guide RNA binding to the Bxb1 attP integrase attachment site, and characterized the detection limits of the system by measuring the minimal small molecule concentration and shortest induction time necessary to produce measurable differences in array composition as read out by Oxford Nanopore sequencing technology.
Type Conference paper
URL https://www.biorxiv.org/content/10.1101/225151v3
Tag SM19-seed
ID 2019b
Funding ICB Network19
Flags
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