Difference between revisions of "Part:BBa K3254016"

(→‎Thermodynamic Characterization)
(→‎Thermodynamic Characterization)
Line 6: Line 6:
  
 
=Thermodynamic Characterization=
 
=Thermodynamic Characterization=
−
*This part is an improved version of the Ptac promoter ([[Part:BBa_K2572025|BBa_K2572025]]) for getting a lower non-induced and full-induced activity.  
+
*This part was an improved version of the Ptac promoter ([[Part:BBa_K2572025|BBa_K2572025]]) for getting a lower non-induced and full-induced activity.  
−
*We used this part to construct an IPTG inducible transcriptional device by combining with a lacI expression cassette([[Part:BBa_K3254022|BBa_K3254022]]) on a P15A plamisd. An insulated sfgfp reporter translational unit ([[Part:BBa_K3254024|BBa_K3254024]]) was applied for quantitative assay the dynamic response curve. The full structure of this operon is similar to [[Part:BBa_K3254025|BBa_K3254025]].
+
*We used this part to construct an IPTG inducible transcriptional device by combining with a lacI expression cassette([[Part:BBa_K3254022|BBa_K3254022]]) on a P15A plamisd. An insulated sfgfp reporter translational unit ([[Part:BBa_K3254024|BBa_K3254024]]) was applied for quantitative assay the dynamic response curve. The full structure of this operon was similar to [[Part:BBa_K3254025|BBa_K3254025]].
−
*The series of IPTG inducible promoters with different dynamic ranges and non-induced activities include [[Part:BBa_K864400|Original Ptac]], [[Part:BBa_K2572025|Ptac]], [[Part:BBa_K3254015|Ptac-M1]], [[Part:BBa_K3254016|Ptac-M2]] and [[Part:BBa_K3254017|Ptac-M3]].
+
*The series of IPTG inducible promoters with different dynamic ranges and non-induced activities included [[Part:BBa_K864400|Original Ptac]], [[Part:BBa_K2572025|Ptac]], [[Part:BBa_K3254015|Ptac-M1]], [[Part:BBa_K3254016|Ptac-M2]] and [[Part:BBa_K3254017|Ptac-M3]].
  
 
==Genetic Design==
 
==Genetic Design==
 
[[File:T--GENAS_China--Ptac-sfGFP_plasmid.PNG|200px|thumb|left|The structure of the experimental plasmid]]
 
[[File:T--GENAS_China--Ptac-sfGFP_plasmid.PNG|200px|thumb|left|The structure of the experimental plasmid]]
−
*The sequence of [[Part:BBa_K3254025|BBa_K3254025]] can be seen as a reference.
+
*The sequence of [[Part:BBa_K3254025|BBa_K3254025]] could be seen as a reference.
−
*The host cell is E.coli DH5α.
+
*The host cell was E.coli DH5α.
  
 
==Experimental Setup==
 
==Experimental Setup==
−
*All incubations were carried out using a Digital Thermostatic Shaker maintained at 37 °C and 1000 rpm, using Corning flat-bottom 96-well plates sealed with sealing film. For characterization the circuit response functions, a previously developed quantitative method that measures gene expression at steady state was used(Zhang, Chen et al. 2016). Briefly, bacteria harboring the parts/circuits of interest were first inoculated from single colonies into a flat-bottom 96-well plate for overnight growth, after which the cell cultures were diluted 196-fold with M9 medium. After 3 h of growth, the cultures were further diluted 700-fold with M9 medium containing gradient concentrations of IPTG, and incubated for another 6 h. Finally, 20-μL samples of each culture were transferred to a new plate containing 180 μL per well of PBS supplemented with 2 mg/mL kanamycin to terminate protein expression. The fluorescence distribution of each sample was assayed using a flow cytometer with appropriate voltage settings; each distribution contained >20,000 events. Each sample was experimentally assayed at least three times. The arithmetical mean of each sample was determined using FlowJo software.
+
*All incubations were carried out using a Digital Thermostatic Shaker maintained at 37 °C and 1000 rpm, using Corning flat-bottom 96-well plates sealed with sealing film. For characterization the circuit response functions, a previously developed quantitative method that measured gene expression at steady state was used(Zhang, Chen et al. 2016). Briefly, bacteria harboring the parts/circuits of interest were first inoculated from single colonies into a flat-bottom 96-well plate for overnight growth, after which the cell cultures were diluted 196-fold with M9 medium. After 3 h of growth, the cultures were further diluted 700-fold with M9 medium containing gradient concentrations of IPTG, and incubated for another 6 h. Finally, 20-μL samples of each culture were transferred to a new plate containing 180 μL per well of PBS supplemented with 2 mg/mL kanamycin to terminate protein expression. The fluorescence distribution of each sample was assayed using a flow cytometer with appropriate voltage settings; each distribution contained >20,000 events. Each sample was experimentally assayed at least three times. The arithmetical mean of each sample was determined using FlowJo software.
 
*M9 medium (supplemented）: 6.8 g/L Na<sub>2</sub>HPO<sub>4</sub>, 3 g/L KH<sub>2</sub>PO<sub>4</sub>, 0.5 g/L NaCl, 1 g/L NH<sub>4</sub>Cl, 0.34 g/L thiamine, 0.2% casamino acids, 0.4% glucose, 2mM MgSO<sub>4</sub>, and 100 μM CaCl<sub>2</sub>.
 
*M9 medium (supplemented）: 6.8 g/L Na<sub>2</sub>HPO<sub>4</sub>, 3 g/L KH<sub>2</sub>PO<sub>4</sub>, 0.5 g/L NaCl, 1 g/L NH<sub>4</sub>Cl, 0.34 g/L thiamine, 0.2% casamino acids, 0.4% glucose, 2mM MgSO<sub>4</sub>, and 100 μM CaCl<sub>2</sub>.
  
 
==Results==
 
==Results==
−
*Those promoters have gentle response curves without saltus.
+
*Those promoters had gentle response curves without saltus.
 
[[File:T--GENAS_China--IPTG-sfGFP.PNG]]
 
[[File:T--GENAS_China--IPTG-sfGFP.PNG]]
 
*Background: Cells without any FP genes.
 
*Background: Cells without any FP genes.

Revision as of 11:13, 21 October 2019


Mutant Ptac promoter No.2

This is a mutant Ptac promoter based on the BBa_K2572025. The -10 region was mutated to GATAAT from TATAAT, and has an "ideal" lac operator. This promoter has a lower leakage.

Thermodynamic Characterization

  • This part was an improved version of the Ptac promoter (BBa_K2572025) for getting a lower non-induced and full-induced activity.
  • We used this part to construct an IPTG inducible transcriptional device by combining with a lacI expression cassette(BBa_K3254022) on a P15A plamisd. An insulated sfgfp reporter translational unit (BBa_K3254024) was applied for quantitative assay the dynamic response curve. The full structure of this operon was similar to BBa_K3254025.
  • The series of IPTG inducible promoters with different dynamic ranges and non-induced activities included Original Ptac, Ptac, Ptac-M1, Ptac-M2 and Ptac-M3.

Genetic Design

The structure of the experimental plasmid
  • The sequence of BBa_K3254025 could be seen as a reference.
  • The host cell was E.coli DH5α.

Experimental Setup

  • All incubations were carried out using a Digital Thermostatic Shaker maintained at 37 °C and 1000 rpm, using Corning flat-bottom 96-well plates sealed with sealing film. For characterization the circuit response functions, a previously developed quantitative method that measured gene expression at steady state was used(Zhang, Chen et al. 2016). Briefly, bacteria harboring the parts/circuits of interest were first inoculated from single colonies into a flat-bottom 96-well plate for overnight growth, after which the cell cultures were diluted 196-fold with M9 medium. After 3 h of growth, the cultures were further diluted 700-fold with M9 medium containing gradient concentrations of IPTG, and incubated for another 6 h. Finally, 20-μL samples of each culture were transferred to a new plate containing 180 μL per well of PBS supplemented with 2 mg/mL kanamycin to terminate protein expression. The fluorescence distribution of each sample was assayed using a flow cytometer with appropriate voltage settings; each distribution contained >20,000 events. Each sample was experimentally assayed at least three times. The arithmetical mean of each sample was determined using FlowJo software.
  • M9 medium (supplemented）: 6.8 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 0.34 g/L thiamine, 0.2% casamino acids, 0.4% glucose, 2mM MgSO4, and 100 μM CaCl2.

Results

  • Those promoters had gentle response curves without saltus.

T--GENAS China--IPTG-sfGFP.PNG

References

  • Zhang HM, et al. Measurements of Gene Expression at Steady State Improve the Predictability of Part Assembly. ACS Synthetic Biology 5, 269-273 (2016).
  • Zong Y, et al. Insulated transcriptional elements enable precise design of genetic circuits. Nature communications 8, 52 (2017).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]