Revision as of 13:30, 21 October 2019

Optimized Ptac promoter

This part is a twin of BBa_K2572025, we built this page just for more detailed sequence annotations. This promoter was optimized from Ptac promoter(BBa_K864400) through using a symmetrical lacO site. After combined with the lacI constitutive expression cassette(BBa_K3254022), it can be regulated by the IPTG inducer. A mutant library with different dynamic range had been built(see BBa_K3254015,BBa_K3254016 and BBa_K3254017).

Thermodynamic Characterization

• You can find the same contents below on the page of BBa_K2572025.
• This part can be seen as an improved version of the original Ptac promoter (BBa_K864400). The main improvement point was this version could be applied a symmetrical lacO site to get a more tightly regulation.
• The page of a twin part was build for a full annotation (BBa_K3254014).
• We used this part to construct an IPTG inducible transcriptional device by combining with a lacI expression cassette(BBa_K3254022) on a P15A plasmid. An insulated sfgfp reporter translational unit (BBa_K3254024) was applied for quantitative assay the dynamic response curve. The full structure of this operon was described on the page of BBa_K3254025.
• Furthermore, we improved this part by mutant the -10 region of this promoter and successfully achieved a series of IPTG inducible promoters(Ptac-M1, Ptac-M2, Ptac-M3) with different dynamic ranges and lower non-induced activities.

Genetic Design

The structure of the experimental plasmid

• The sequence detail was described on the page of BBa_K3254025.
• 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 characterizing 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 have 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 BBa_K3254014 Sequence And Features Not understood