Difference between revisions of "Part:BBa K3712015"
 
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===RESULTS===
 
===RESULTS===
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<p>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.</p>
 
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<center><img src="https://2021.igem.org/wiki/images/9/9d/T--HUST2-China--SDS-PAGE_of_Wild-type_BLP-7-27_ELP%2C_Optimized_BLP-7-27_ELP%2C_Wild-type_TLR2-27ELP%2C_Optimized_TLR2-27_ELP_expression_results.jpg"） style="width:793px;height:360px"></center>
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<p><b>Figure 1.</b>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.
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<p><b>Figure 1</b> The agarose gel electrophoresis map of Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP, Wild-type TLR2-27ELP, and Optimized TLR2-27 ELP respectively constructed in pR/pL expression system. About 200-1000 ng of plasmid was digested at 37℃ for 30-60 minutes and analyzed on 1% Agarose Gel. Lane1: circular plasmid (brighter) and supercoiled structure; Lane2: plasmid (brighter) digested by EcoRI and BamHI and target gene fragment. </p>
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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.
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<p><b>Figure 2.</b>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..
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<h1>Future direction:</h1>
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<p>(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.
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(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.
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Latest revision as of 13:58, 21 October 2021


pBV220 plasmid

USAGE AND BIOLOGY

The SD sequence of pBV220 plasmid (sequence used to bind prokaryotic ribosomes) is followed by a polyclonal restriction site, which is convenient for inserting foreign genes with the starting ATG, and can express non-fusion proteins.The vector contains a strong transcription terminator to prevent the "read through" phenomenon, which is conducive to the stability of the plasmid-host system.The vector pBV220 is 3.7Kb and helps increase its copy number and capacity. pBV220 contains the PL promoter and the cI protein gene cIts857, which codes for the inhibitory effect of the promoter and is temperature-sensitive, to regulate the gene pL/pR, so the transcription of the foreign gene inserted into it can be regulated by temperature.

RESULTS

Part:BBa_K3712013

Figure 1 The agarose gel electrophoresis map of Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP, Wild-type TLR2-27ELP, and Optimized TLR2-27 ELP respectively constructed in pR/pL expression system. About 200-1000 ng of plasmid was digested at 37℃ for 30-60 minutes and analyzed on 1% Agarose Gel. Lane1: circular plasmid (brighter) and supercoiled structure; Lane2: plasmid (brighter) digested by EcoRI and BamHI and target gene fragment.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1
    Illegal PstI site found at 40
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1
    Illegal PstI site found at 40
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1
    Illegal BglII site found at 3419
    Illegal BamHI site found at 7
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1
    Illegal PstI site found at 40
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1
    Illegal PstI site found at 40
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 220
    Illegal BsaI site found at 1287
    Illegal SapI.rc site found at 2369