Part:BBa_K3117030:Experience

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Applications of BBa_K3117030

Usage of the composite part by the iGEM team FAU_Erlangen:

Construct 2b (K2b) was synthesized by IDT. 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.

To assure DNA was correctly inserted during cloning samples were sequenced. Data is depicted in Fig. 1.

Figure 1: Sequencing data of K2b

Fig. 2 shows the harvest after transfection of HEK 293T cells in a western blot. For detection, the His-Tag (BBa_K3117005) in the K2b sequence provides the opportunity to be used as a target for a primary antibody in a western blot. K2b can be seen at expected height (41 kDa). Presence of K2b in the medium proves the function of the Igk leader (BBa_K3117006), which is directing the protein into the secretory pathway.

Figure 2: Western blot of the harvest after transfection into HEK 293T cells with an anti His-Tag antibody

Furthermore the His-Tag (BBa_K3117006) in K2b allows purification of the protein with HisTrap columns. The western blot after this purification is shown in Fig. 3, showing the remaining presence of the protein after this step.

Figure 3: Western blot of the constructs after the purification via HisTrap

For the SpyTag/SpyCatcher reaction the protein K2b was added to the protein K2a (BBa_K3117026). 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 a trimeric protein (Schoene, Fierer, Bennett, & Howarth, 2014).

Figure 4: Western blot of the constructs K2a, K2b and K2a+b (1:1 ratio K2a with K2b) with an anti His-Tag antibody

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