Latest revision as of 11:45, 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 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.
• 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.

• Background: Cells without any FP genes.
• Ptac with normal lacO: BBa_K864400
• Ptac with symmetrical lacO: BBa_K2572025
• Ptac-M1: BBa_K3254015
• Ptac-M2: BBa_K3254016
• Ptac-M3: BBa_K3254017

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