Proof of concept continuous event logging in living cells
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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 such outside stimuli. In this work, we have engineered a genetic circuit that can enact specific and controlled genetic changes in response to changing small molecule concentrations. Known DNA sequences can be repeatedly integrated into 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 long read sequencing technology.
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SciScore for 10.1101/225151: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication Authentication: Genome site constructs were made by Gibson assembly into SpeI-KpnI digested pOSIP KH or pOSIP KO from Pierre et al [19], followed by genome integration and pE-FLP excision protocol as described. Table 2: Resources
Recombinant DNA Sentences Resources Genome site constructs were made by Gibson assembly into SpeI-KpnI digested pOSIP KH or pOSIP KO from Pierre et al [19], followed by genome integration and pE-FLP excision protocol as described. pE-FLPsuggested: RRID:Addgene_45978)dCas9 was amplified from pAN-PTet-dCas9, which was a gift from … SciScore for 10.1101/225151: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication Authentication: Genome site constructs were made by Gibson assembly into SpeI-KpnI digested pOSIP KH or pOSIP KO from Pierre et al [19], followed by genome integration and pE-FLP excision protocol as described. Table 2: Resources
Recombinant DNA Sentences Resources Genome site constructs were made by Gibson assembly into SpeI-KpnI digested pOSIP KH or pOSIP KO from Pierre et al [19], followed by genome integration and pE-FLP excision protocol as described. pE-FLPsuggested: RRID:Addgene_45978)dCas9 was amplified from pAN-PTet-dCas9, which was a gift from Christopher Voigt (Addgene plasmid # 62244) [20]. pAN-PTet-dCas9suggested: RRID:Addgene_62244)n, CinR, and pBAD were amplified from source material with primers to add BsaI cut sites using compatible sequences [21] before being used for assembly. pBADsuggested: RRID:Addgene_105665)Sequencing: PCR products for pF1 constructs were obtained using pASS_pF1F: aaaCACCTGCaaaaTTACGGCGTATCACGAGGCAGAAT and pASS_genR: aaaCACCTGCaaaaCCTGGTACAGACAGGAGCTGCGTT. pF1suggested: RRID:Addgene_34515)PCR products for pF2 constructs were obtained with pASS_pF2F: aaaCACCTGCaaaaTTACCGGTATCAACAGGGACACC and pASS_genR as above. pF2suggested: RRID:Addgene_42520)pASS_genRsuggested: NoneSoftware and Algorithms Sentences Resources Loading on MinION R9.4 flow cell proceeded as per manufacturer recommendations. MinIONsuggested: (MinION, RRID:SCR_017985)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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