Difference between revisions of "Part:BBa K2924032"
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===Usage and Biology===
 
===Usage and Biology===
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[[File:PET22b_as1casein.png|500px|thumb|right|<html><i>Fig. 1: Scheme of α-s1-casein overexpression construct. The insert, containing the promoter P<sub>T7</sub> (<a href="https://parts.igem.org/Part:BBa_K2406020">BBa_K2406020</a>), α-s1-casein gene (<a href="https://parts.igem.org/Part:BBa_K2924026">BBa_K2924026</a>) fused with a 6x His-tag and the T7 terminator (<a href="https://parts.igem.org/Part:BBa_B0012">BBa_B0012</a>), was cloned into the pET22b backbone</i></html>]]
  
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Using the strong T7 expression system, the P<sub>T7</sub> promoter (BBa_K2406020) expresses α-s1-casein (BBa_K2924026) with the T7 terminator (BBa_B0012). The transcription of the T7 promoter is carried out by the T7-RNA polymerase, whose expression is inhibited by <i>lacI</i> and can be induced by either lactose or IPTG<sup>1</sup>. The lac-operator is also present between the promoter and the gene to reduce the leakiness of the expression by requiring derepression of the T7-RNA polymerase as well as the target gene’s expression. In addition the used pET22b contains the Ribosome-binding site (BBa_K2924053)<sup>2</sup>.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2924032 parameters</partinfo>
 
<partinfo>BBa_K2924032 parameters</partinfo>
 
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===Characterization===
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α-s1-casein was cloned into the T7 promoter-based vector pET22b which is a high copy plasmid and contains the pelB signal sequence, a specific amino acid sequence that is fused to the protein and facilitates the export into the periplasm. The plasmid contains a fused histidine tag (6xHis) for easier purification of overexpressed proteins.
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The strain BL21(DE3) was used for expression. This strain is a common strain for expression because it contains T7 polymerase in its genome and expresses fewer proteases, which might degrade the product<sup>3</sup>. For the IPTG-induced overexpression 100 ml LB containing Ampicillin [100mg/ml] were inoculated with 1 ml of overnight culture.
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The cells were incubated at 37° C at 180 rpm until an OD600 of 0.6 was reached. <a href="https://www.protocols.io/view/iptg-induced-overexpression-in-e-coli-8ffhtjn">IPTG</a> [0.5 mM] was added and the cultures were incubated for another 2 h at 37° C at 180 rpm. The cultures were centrifuged at 8000 x g  for 20 min at 4°C to separate the cell pellet from the medium, both were kept on ice. The cell pellet was lysed with a lysis reagent. The samples were used for <a href="https://www.protocols.io/view/sds-page-8evhte6">Sodium dodecyl sulfate polyacrylamide gel electrophoresis</a>  (SDS-PAGE) to prove the heterologous expressed α-s1-casein which has a size of approximately 25.4 kDa size (Fig. 2).

Revision as of 22:33, 19 October 2019


T7 + RBS + α-s1-casein + terminator

T7-lacO promoter (BBa_K2406020) and RBS expressing α-s1-casein (BBa_K2924026) with 6xHis-tag and T7-Terminator (BBa_B0012) in E. coli

Usage and Biology

Fig. 1: Scheme of α-s1-casein overexpression construct. The insert, containing the promoter PT7 (BBa_K2406020), α-s1-casein gene (BBa_K2924026) fused with a 6x His-tag and the T7 terminator (BBa_B0012), was cloned into the pET22b backbone

Using the strong T7 expression system, the PT7 promoter (BBa_K2406020) expresses α-s1-casein (BBa_K2924026) with the T7 terminator (BBa_B0012). The transcription of the T7 promoter is carried out by the T7-RNA polymerase, whose expression is inhibited by lacI and can be induced by either lactose or IPTG1. The lac-operator is also present between the promoter and the gene to reduce the leakiness of the expression by requiring derepression of the T7-RNA polymerase as well as the target gene’s expression. In addition the used pET22b contains the Ribosome-binding site (BBa_K2924053)2. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 756
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

α-s1-casein was cloned into the T7 promoter-based vector pET22b which is a high copy plasmid and contains the pelB signal sequence, a specific amino acid sequence that is fused to the protein and facilitates the export into the periplasm. The plasmid contains a fused histidine tag (6xHis) for easier purification of overexpressed proteins.

The strain BL21(DE3) was used for expression. This strain is a common strain for expression because it contains T7 polymerase in its genome and expresses fewer proteases, which might degrade the product3. For the IPTG-induced overexpression 100 ml LB containing Ampicillin [100mg/ml] were inoculated with 1 ml of overnight culture.

The cells were incubated at 37° C at 180 rpm until an OD600 of 0.6 was reached. IPTG [0.5 mM] was added and the cultures were incubated for another 2 h at 37° C at 180 rpm. The cultures were centrifuged at 8000 x g for 20 min at 4°C to separate the cell pellet from the medium, both were kept on ice. The cell pellet was lysed with a lysis reagent. The samples were used for Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to prove the heterologous expressed α-s1-casein which has a size of approximately 25.4 kDa size (Fig. 2).