Part:BBa_K652002
T7-RBS-His-eGFP-TEV-TmAFP-Term
This part contains a T7 strong promoter, RBS, our eGFP-fusion protein containing the Tenebrio molitor antifreeze protein (TmAFP) with a His tag at the N-terminus, followed by a terminator.
More information about this and other parts can be found on our wiki under the project section: http://2011.igem.org/Team:Yale
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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 897
Illegal AgeI site found at 936
Illegal AgeI site found at 1008 - 1000COMPATIBLE WITH RFC[1000]
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