Difference between revisions of "Part:BBa K3117026:Experience"

Revision as of 17:16, 21 October 2019

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Applications of BBa_K3117026

Use of the part by the iGEM Team FAU_Erlangen:

The construct 2a (K2a) was synthesized by IDT. Therefore, it was cloned into the expression vector pSEC/tag2/Hygro\C\-\OK by Gibson Assembly. Our protein was under control of the CMV promotor ensuring a high-level constitutive expression. Afterwards, the construct was brought into HEK293T cells with either calcium phosphate transfection or lipofection.

Accuracy of the inserted DNA after cloning was confirmed by sequencing. The data is depicted in Fig. 1.

Bild Sequencing K2a ![Figure 1: Sequencing data of K2a](/path/name)

Fig.2 shows the western blot of the harvest after transfection into HEK 293T cells. For detection, the His-Tag in the K2a sequence (BBa_K3117005) provides the opportunity to be used as a target for a primary antibody in a western blot. K2a can be seen at expected height (42 kDa). Blurry bands might resulted from protease degradation. The presence of K2a in the medium proves the function of the Igk leader (BBa_K3117006), which is directing the protein into the secretory pathway.

Bild Ernte ![Figure 2: Western blot of the harvest after transfection into HEK 293T cells with an anti-His-Tag antibody](/path/name)

Furthermore the His-Tag (BBa_K3117005) in K2a allows purification of the protein with HisTrap columns. The western blot after purification is shown in Fig. 3, depicting the remaining presence of the protein after this step.

Bild Aufreinigung ![Figure 3: Western blot of the purified protein with HisTrap columns with an anti-His-Tag antibody](/path/name)

For the SpyTag/SpyCatcher reaction the protein K2a was added to the protein K2b (BBa_K3117030). A height of 83 kDa was expected in the afterwards performed western blot, depicted in Fig 4. A specific band appeared after adding the two products, but this band was running at ~120 kDa. This size might be explained by formation of trimeric protein (Schoene, Fierer, Bennett, & Howarth, 2014).

Bild Tag/Catcher ![Figure 4: Western blot after SpyTag/SpyCatcher reaction](/path/name)

User Reviews

UNIQ7e366635779c373c-partinfo-00000000-QINU UNIQ7e366635779c373c-partinfo-00000001-QINU

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 ===Applications of BBa_K3117026===
-Use of the part by the iGEM Team Erlangen:
+Use of the part by the iGEM Team FAU_Erlangen:
 The construct 2a (K2a) was synthesized by IDT. Therefore, it was cloned into the expression vector pSEC/tag2/Hygro\C\-\OK by Gibson Assembly. Our protein was under control of the CMV promotor ensuring a high-level constitutive expression. Afterwards, the construct was brought into HEK293T cells with either calcium phosphate transfection or lipofection.