Part:BBa_K193209:Experience

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Applications of BBa_K193209

Team Yale has submitted a sequence of this biobrick that is BBF RFC10 compatible (BBa_K652002 and BBa_K652003). See: https://parts.igem.org/wiki/index.php?title=Part:BBa_K652002.

Our biobrick contains a T7 strong promoter, RBC, eGFP, followed by the Tenebrio molitor antifreeze protein (TmAFP) with a His tag at the N-terminus, followed by a terminator. Unlike the Tokyo Tech TmAFP biobrick, ours does not contain an internal EcoRI restriction site. Additionally, BBa_K652002 has a TEV protease linker between the eGFP and the TmAFP which allows for isolation of the TmAFP part.

We kindly obtained the sequence of the TmAFP from the Fass Lab, reported on in the following paper:

Bar, M., Bar-Ziv, R., Scherf, T. & Fass, D. Efficient production of a folded and functional, highly disulfide-bonded [beta]-helix antifreeze protein in bacteria. Protein Expression and Purification 48, 243-252 (2006).

The sequence of the Tokyo Tech part BBa_K193209 seems to have part of the TmAFP protein missing. Additionally, it does not have a terminator. Our sequence does not have these problems.

In general, the TmAFP proteins do not express very well in E. coli, even if the Origami 2 strain is used. This is because the protein is highly disulfided. Most of the protein ends up in inactive insoluble form in the pellet (we detected this using SDS-PAGE and using a Western Blot). We used fluorimetry to prove that there was some properly folded eGFP-TmAFP, but based on our purified eGFP standard this is likely in the micromolar quantity. UV-vis was not sensitive enough to detect the concentration of eGFP-TmAFP. And of course, the pellet was not green :(.

Figure 1: Western Blot, probed with mouse α-GFP-IgG2a antibody. Lanes 0: Ladder. Lane 9: uninduced eGFP-TEV-TmAFP. Lane 10: induced eGFP-TEV-TmAFP. Lane 11: Ladder.

Figure 2: Western Blot, probed with His antibody; uninduced eGFP-TEV-TmAFP (left), induced eGFP-TEV-TmAFP (right)

Figure 3: SDS-PAGE, probed with Coomassie blue; uninduced eGFP-TEV-TmAFP (left), induced eGFP-TEV-TmAFP (right)

Figure 4: Fluorimetry was used to measure flourescence. Excitation wavelength 488nm. Emission scan 500 to 650 nm

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