Difference between revisions of "Part:BBa K2924032"

Latest revision as of 19:06, 21 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 P_T7 (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 P_T7 expresses α-s1-casein with the T7 terminator. 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 . </html> Sequence and Features

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.

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Fig. 2: SDS-PAGE of E.coli protein. The lysed cells of E.coli BL21 with pET22b+α-s1-casein and the empty vector control were applied. The SDS-PAGE was run at 220V, 400 A for 45 minutes and then stained with a Coomassie blue dye. α-s1-casein has an expected size of 25.4 kDa

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).

Fig. 2 shows a band of almost 25 kDa in the cell pellet sample of E.coli BL21 with α-s1-casein, there is no band visible in comparison to the empty vector control of E.coli. This band is very likely the α-s1-casein protein, which ran slightly lower than expected. The band is faint compared to most other protein bands on the gel, suggesting that the yield is low for this expression. Additionally there is a new band visible around 55 kDa, which is not present in the empty vector, which might be a dimer of α-s1-casein, since it has roughly twice the mass of a monomer.

The results indicate, that E. coli could be suitable for the production of milk proteins, however, further experiments need to be carried out to observe the secretion efficiency for the protein and to determine how much of the protein can be produced.

These results confirm the functionality and successful implementation of our basic concept of repressing specific genes.

References

1: Tabor, Stanley. "Expression using the T7 RNA polymerase/promoter system." Current protocols in molecular biology 11.1 (1990): 16-2.

2: Dubendorf, John W., and F. William Studier. "Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor." Journal of molecular biology 219.1 (1991): 45-59.