2 research outputs found
Biophysical modeling of bacterial restriction-modification systems
Π Π΅ΡΡΡΠΈΠΊΡΠΈΠΎΠ½ΠΎ-ΠΌΠΎΠ΄ΠΈΡΠΈΠΊΠ°ΡΠΈΠΎΠ½ΠΈ (Π -Π) ΠΈ CRISPR-Cas ΡΠΈΡΡΠ΅ΠΌΠΈ ΠΊΠΎΡΠΈΡΡΠ΅ ΡΠ°Π·Π»ΠΈΡΠΈΡΠ΅ ΠΌΠ΅Ρ
Π°Π½ΠΈΠ·ΠΌΠ΅ Π·Π° ΠΎΠ±Π°Π²ΡΠ°ΡΠ΅ ΠΎΡΠ½ΠΎΠ²Π½Π΅ ΡΡΠ½ΠΊΡΠΈΡΠ΅ β ΠΎΠ΄Π±ΡΠ°Π½Π΅ ΠΏΡΠΎΠΊΠ°ΡΠΈΠΎΡΡΠΊΠ΅ ΡΠ΅Π»ΠΈΡΠ΅ ΠΎΠ΄ ΡΡΡΠ°Π½Π΅ ΠΠΠ. ΠΠ° ΡΠ΅ΡΠΈΡΠΈ ΠΎΠ΄Π°Π±ΡΠ°Π½Π° Π -Π ΡΠΈΡΡΠ΅ΠΌΠ° Π’ΠΈΠΏΠ° II ΠΈ CRISPR-Cas Π’ΠΈΠΏΠ° I-E ΡΡ ΠΏΠΎΡΡΠ°Π²ΡΠ΅Π½ΠΈ ΡΠ΅ΡΠΌΠΎΠ΄ΠΈΠ½Π°ΠΌΠΈΡΠΊΠΈ ΠΌΠΎΠ΄Π΅Π»ΠΈ ΡΠ΅Π³ΡΠ»Π°ΡΠΈΡΠ΅ ΡΡΠ°Π½ΡΠΊΡΠΈΠΏΡΠΈΡΠ΅ ΠΈ Π΄ΠΈΠ½Π°ΠΌΠΈΡΠΊΠΈ ΠΌΠΎΠ΄Π΅Π»ΠΈ Π΅ΠΊΡΠΏΡΠ΅ΡΠΈΡΠ΅ ΡΡΠ°Π½ΡΠΊΡΠΈΠΏΠ°ΡΠ° ΠΈ ΠΏΡΠΎΡΠ΅ΠΈΠ½Π°. Π‘ΠΈΠΌΡΠ»Π°ΡΠΈΡΠΎΠΌ ΠΈ Π°Π½Π°Π»ΠΈΠ·ΠΎΠΌ Π΄ΠΈΠ½Π°ΠΌΠΈΠΊΠ΅ ΠΌΠΎΠ΄Π΅Π»Π° ΡΡ ΠΈΠ΄Π΅Π½ΡΠΈΡΠΈΠΊΠΎΠ²Π°Π½Π΅ ΠΎΡΠΎΠ±ΠΈΠ½Π΅ Π΄ΠΈΠ½Π°ΠΌΠΈΠΊΠ΅ Π΅ΠΊΡΠΏΡΠ΅ΡΠΈΡΠ΅ ΡΠΈΡΡΠ΅ΠΌΠ° ΠΏΠΎ ΠΏΠΎΠΊΡΠ΅ΡΠ°ΡΡ ΡΠΈΡ
ΠΎΠ²Π΅ Π°ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ Ρ ΡΠ΅Π»ΠΈΡΠΈ ΠΊΠΎΡΠ΅ Π²Π΅ΡΠΎΠ²Π°ΡΠ½ΠΎ ΠΏΡΠ΅Π΄ΡΡΠ°Π²ΡΠ°ΡΡ ΠΏΡΠΈΠ½ΡΠΈΠΏΠ΅ Π΅Π²ΠΎΠ»ΡΡΠΈΠ²Π½ΠΎΠ³ Π΄ΠΈΠ·Π°ΡΠ½Π° ΡΠΈΡ
ΠΎΠ²Π΅ ΡΠ΅Π³ΡΠ»Π°ΡΠΈΡΠ΅. ΠΡΠ΅ΡΠΈΠ·Π½ΠΈΡΠ΅, ΠΈΡΠΏΠΈΡΠ°Π½ΠΎ ΡΠ΅: i) ΠΊΠ°ΠΊΠΎ ΠΏΠ΅ΡΡΡΡΠ±Π°ΡΠΈΡΠ΅ ΠΊΠ°ΡΠ°ΠΊΡΠ΅ΡΠΈΡΡΠΈΡΠ½ΠΈΡ
ΡΠ΅Π³ΡΠ»Π°ΡΠΎΡΠ½ΠΈΡ
ΡΠ²ΠΎΡΡΡΠ°Π²Π° Π -Π ΡΠΈΡΡΠ΅ΠΌΠ° AhdI ΠΈ EcoRV ΡΡΠΈΡΡ Π½Π° ΡΡΠΈ ΠΏΡΠ΅Π΄Π»ΠΎΠΆΠ΅Π½Π° Π΄ΠΈΠ½Π°ΠΌΠΈΡΠΊΠ° ΠΏΡΠΈΠ½ΡΠΈΠΏΠ°; ii) Π΄Π° Π»ΠΈ Π -Π ΡΠΈΡΡΠ΅ΠΌ Kpn2I, ΡΠ° ΡΠ΅Π³ΡΠ»Π°ΡΠΈΡΠΎΠΌ Π½Π° Π½ΠΈΠ²ΠΎΡ Π΅Π»ΠΎΠ½Π³Π°ΡΠΈΡΠ΅ ΡΡΠ°Π½ΡΠΊΡΠΈΠΏΡΠΈΡΠ΅, ΠΌΠΎΠΆΠ΅ Π΄Π° ΠΎΠ±Π΅Π·Π±Π΅Π΄ΠΈ ΠΎΡΠ΅ΠΊΠΈΠ²Π°Π½Π° Π΄ΠΈΠ½Π°ΠΌΠΈΡΠΊΠ° ΡΠ²ΠΎΡΡΡΠ²Π°; iii) Π΄Π° Π»ΠΈ ΡΡ ΠΏΠΎΡΡΠΎΡΠ΅ΡΠ° ΡΠ°Π·Π½Π°ΡΠ° ΠΎ ΡΠ΅Π³ΡΠ»Π°ΡΠΈΡΠΈ Π -Π ΡΠΈΡΡΠ΅ΠΌΠ° Esp1396I Π΄ΠΎΠ²ΠΎΡΠ½Π° Π·Π° ΡΠ΅ΠΏΡΠΎΠ΄ΡΠΊΠΎΠ²Π°ΡΠ΅ Π΄ΠΈΠ½Π°ΠΌΠΈΠΊΠ΅ Π΅ΠΊΡΠΏΡΠ΅ΡΠΈΡΠ΅ ΠΏΡΠΎΡΠ΅ΠΈΠ½Π° ΠΈΠ·ΠΌΠ΅ΡΠ΅Π½Π΅ Π½Π° Π½ΠΈΠ²ΠΎΡ ΠΏΠΎΡΠ΅Π΄ΠΈΠ½Π°ΡΠ½ΠΈΡ
ΡΠ΅Π»ΠΈΡΠ°; iv) ΠΊΠ°ΠΊΠ²Π΅ ΠΎΡΠΎΠ±ΠΈΠ½Π΅ Π²Π΅ΡΠΎΠ²Π°ΡΠ½ΠΎ ΠΈΠΌΠ° Π½Π΅ΠΏΠΎΠ·Π½Π°ΡΠ° Π΄ΠΈΠ½Π°ΠΌΠΈΠΊΠ° Π΅ΠΊΡΠΏΡΠ΅ΡΠΈΡΠ΅ CRISPR-Cas ΡΠΈΡΡΠ΅ΠΌΠ° Ρ Escherichia coli, ΠΏΡΠ΅Π΄Π²ΠΈΡΠ΅Π½Π° ΡΠ· ΠΏΡΠ΅ΡΠΏΠΎΡΡΠ°Π²ΠΊΡ Π΄Π° ΡΠ΅ ΡΠ΅Π³ΠΎΠ² ΠΌΠ΅Ρ
Π°Π½ΠΈΠ·Π°ΠΌ ΡΠ΅Π³ΡΠ»Π°ΡΠΈΡΠ΅ ΡΡΠ°Π½ΡΠΊΡΠΈΠΏΡΠΈΡΠ΅ ΠΌΠΎΠΆΠ΅ Π°ΠΏΡΠΎΠΊΡΠΈΠΌΠΈΡΠ°ΡΠΈ ΠΊΠΎΠ½ΡΠ΅ΠΏΡΡΠ°Π»Π½ΠΎ ΡΠ»ΠΈΡΠ½ΠΈΠΌ ΠΌΠ΅Ρ
Π°Π½ΠΈΠ·ΠΌΠΎΠΌ Π -Π ΡΠΈΡΡΠ΅ΠΌΠ°. ΠΠΎΠΊΠ°Π·Π°Π½ΠΎ ΡΠ΅ Π΄Π° ΡΠ²Π° ΡΠ΅ΡΠΈΡΠΈ Π -Π ΡΠΈΡΡΠ΅ΠΌΠ°, ΠΊΠ°ΠΎ ΠΈ CRISPR-Cas, ΡΡΡΡΠΊΡΡΡΠ½ΠΎ ΠΈΡΠΏΡΡΠ°Π²Π°ΡΡ ΡΡΠ»ΠΎΠ²Π΅ Π·Π° ΠΏΠΎΡΡΠΈΠ·Π°ΡΠ΅ Π΄Π²Π° ΠΏΡΠ΅Π΄Π»ΠΎΠΆΠ΅Π½Π° Π΄ΠΈΠ½Π°ΠΌΠΈΡΠΊΠ° ΠΏΡΠΈΠ½ΡΠΈΠΏΠ° β ΠΏΠΎΡΠ΅ΡΠ½ΠΎ ΠΊΠ°ΡΡΠ΅ΡΠ΅ Π΅ΠΊΡΠΏΡΠ΅ΡΠΈΡΠ΅ ΡΠ΅ΡΡΡΠΈΠΊΡΠΈΠΎΠ½Π΅ Π΅Π½Π΄ΠΎΠ½ΡΠΊΠ»Π΅Π°Π·Π΅ Π·Π° Π΅ΠΊΡΠΏΡΠ΅ΡΠΈΡΠΎΠΌ ΠΌΠ΅ΡΠΈΠ»ΡΡΠ°Π½ΡΡΠ΅ΡΠ°Π·Π΅ ΠΈ ΡΠ΅Π½ Π½Π°Π³Π»ΠΈ ΠΏΠΎΡΠ°ΡΡ ΠΊΠ° ΡΡΠ°ΡΠΈΠΎΠ½Π°ΡΠ½ΠΎΠΌ ΡΡΠ°ΡΡ, Π΄ΠΎΠΊ Π°Π½Π°Π»ΠΈΠ·Π° ΡΠΈΡΡΠ΅ΠΌΠ° AhdI ΠΈ EcoRV ΠΏΠΎΠ΄ΡΠΆΠ°Π²Π° ΠΈ ΡΡΠ΅ΡΠΈ β Π½ΠΈΡΠΊΠ΅ ΡΠ»ΡΠΊΡΡΠ°ΡΠΈΡΠ΅ Ρ ΡΡΠ°ΡΠΈΠΎΠ½Π°ΡΠ½ΠΎΠΌ ΡΡΠ°ΡΡ. ΠΠ²Π° ΡΠ°Π·Π½Π°ΡΠ° ΠΎ Π΄ΠΈΠ·Π°ΡΠ½Ρ ΠΏΡΠΈΡΠΎΠ΄Π½ΠΈΡ
Π³Π΅Π½ΡΠΊΠΈΡ
ΠΌΡΠ΅ΠΆΠ° ΠΎΠΌΠΎΠ³ΡΡΠ°Π²Π°ΡΡ Π±ΠΎΡΠ΅ ΡΠ°Π·ΡΠΌΠ΅Π²Π°ΡΠ΅ Π²Π΅Π·Π΅ ΠΈΠ·ΠΌΠ΅ΡΡ ΡΠΈΡ
ΠΎΠ²Π΅ ΡΡΡΡΠΊΡΡΡΠ΅ ΠΈ ΡΡΠ½ΠΊΡΠΈΡΠ΅ ΠΈ Π΄Π°ΡΡ ΡΠΌΠ΅ΡΠ½ΠΈΡΠ΅ Π·Π° Π΄ΠΈΠ·Π°ΡΠ½ ΡΠΈΠ½ΡΠ΅ΡΠΈΡΠΊΠΈΡ
Π³Π΅Π½ΡΠΊΠΈΡ
ΠΊΠΎΠ»Π°.Restriction-modification (R-M) and CRISPR-Cas systems use different mechanisms to perform their main function - defend prokaryotic cells from foreign DNA. Thermodynamic models of transcription regulation and dynamic models of transcript and protein expression were set for four selected Type II R-M systems and a Type I-E CRISPR-Cas. By simulating and analyzing the model dynamics, we identified the properties of the system expression dynamics upon the induction in a cell which may be the principles of the regulation evolutionary design. Specifically, we examined: i) how perturbing of the characteristic regulatory features of the R-M systems AhdI and EcoRV affects the three proposed dynamic principles; ii) if the R-M system Kpn2I, whith regulation at the level of transcription elongation, can provide the expected dynamic properties; iii) if the known regulation of the R-M system Esp1396I is sufficient to reproduce the protein expression dynamics measured on single-cells; iv) which properties are probably found in the unknown expression dynamics of the CRISPR-Cas system in Escherichia coli, predicted under the assumption that its transcription regulation mechanism can be approximated by a similar one from R-M systems. We showed that all four R-M systems, as well as CRISPR-Cas, are able to achieve the two proposed dynamic principles - initial delay of restriction endonuclease with respect to methyltransferase expression and its rapid increase towards steady-state, while analysis of AhdI and EcoRV adds the third principle - low fluctuations in the steady-state. Gained insights into the design of these natural gene networks provide a better understanding of the relationship between their structure and function, as well as guidelines for the design of synthetic gene circuits
Fluorescent reporter genes and the analysis of bacterial regulatory networks
International audienceThe understanding of the regulatory networks controllingthe adaptation of bacteria to changes in their environment is criticallydependent on the ability to monitor the dynamics of gene expression.Here, we review the use of fluorescent reporter genes for dynamicallyquantifying promoter activity and other quantities characterizing geneexpression. We discuss critical physical and biological parameters in thedesign, development, and use of fluorescent reporter strains. Moreover,we review measurement models that have been proposed to interpretprimary fluorescence data and inference methods for estimating geneexpression profiles from these data. As an illustration of the use of fluorescentreporter strains for analyzing bacterial regulatory networks, weconsider two applications in the model bacterium Escherichia coli insome detail: the joint control of gene expression by global physiologicaleffects and specific regulatory interactions, and the importance of proteinstability for the inference and analysis of transcriptional regulatorynetworks. We conclude by discussing some current trends in the use offluorescent reporter genes