Part:BBa_K3712013
pR/pL promotor
USAGE AND BIOLOGY
PR/PL, a tandem PR and PL promoter from lambda phage, to ensure high-level expression of genes.Our target gene was cloned downstream of the pL and/or pR promoters. When cultured at 37℃, the expression of target gene is inhibited by Tcl and the target protein is hardly expressed. Under the induction of temperature at 45℃ or infrared light which increases temperature above 37℃, Tcl degrades and the inhibition of the promoter releases. So, the expression of target protein is initiated.
RESULTS
we transform the constructed plasmids into E. coli Nissle 1917 to express the target proteins. All transformed E. coli Nissle1917 are cultured at 37℃ for 12 hours and induced at 45℃ for one day. The bacterial that are only cultured at 37℃ for 12 hours without transformation and the bacterial that were transformed without induction were used as the control group. The experimental group is induced at 45℃ for one day after transformation and culturation. The standard curve was made by BCA assay. By determinating the appropriate loading concentration, the loading amount of each channel is controlled to 30μg, and then perform SDS-PAGE verify the expression of our product.
Figure 1.SDS-PAGE of Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP, Wild-type TLR2-27ELP, Optimized TLR2-27 ELP expression results. Lane 1: Near the 25kDa marker, the binds of Wild-type TLR2-27ELP and Optimized TLR2-27 ELP after induction are darker than the expression system without induction. Near the 15kDa marker, the bands of Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP after induction are darker than the expression system without induction, which indicates that our constructed pR/pL temperature control system is able to express our product in E. coli Nissle1917. Western Blot is conducted to further verify that the molecule near the marker is the right target protein we want to express. After trarsmembran, we incubated overnight with the primary antibody against His-tag, and then incubated with the secondary antibody for development. After induction, there are obvious bands, as shown in Figure 3, which further proves the success of vector construction and expression of our target protein.
Figure 2.Western blot results of Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP, Wild-type TLR2-27ELP, and Optimized TLR2-27 ELP. A) In the vicinity of 25kDa, the color of Wild-type TLR2-27ELP, Optimized TLR2-27 ELP after induction and the one without induction have obvious color differences. B) Around 25kDa, the color development of Wild-type TLR2-27ELP, Optimized TLR2-27 ELP after induction and the one without induction is different obviously..
Future direction:
(1) Due to time and guidance issues, we could not make up the Western blot with internal parameters, but we can guarantee it is our target protein. Because after the transformation was successful, we stained with Ponceau red to observe obvious bands. Later, we will complete the Western blot with internal parameters as a control to further illustrate the expression of the four proteins Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP, Wild-type TLR2-27ELP, and Optimized TLR2-27 ELP level.
(2) In order to better verify the aggregation properties of ELP, the bactericidal properties of antimicrobial peptides and the anti-inflammatory effects of antagonists, we will continue to express the products we need in the future to further verify the functions of the proteins we need.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 116
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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