Coding

Part:BBa_K4623010

Designed by: Ruixiao Tan   Group: iGEM23_BNU-China   (2023-10-09)
Revision as of 16:11, 11 October 2023 by Tanruixiao (Talk | contribs)


Twisted Silinker(GFP),The process of protein coiling can be visualized and green fluorescence can be

Usage and Biology

The initial intent of this part was to visualize the conformational changes in Twisted Silinker (BBa_K46009). For more details, please refer to our wiki. The core of this part is GCaMP6m (BBa_K3755007). When Calmodulin (CaM) responds to calcium ions and binds to Calmodulin Binding Peptide (CBP), the spatial structure of GFP becomes more compact, leading to an increase in fluorescence intensity. We aimed to provide a measurable and quantifiable reference for the challenging-to-measure phenomenon of TS spatial structural changes through the variation in GFP fluorescence intensity. However, the Twisted Silinker (GFP) we ultimately obtained did not exhibit differences in fluorescence intensity at different calcium ion concentrations.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1015
    Illegal EcoRI site found at 1543
    Illegal EcoRI site found at 1711
    Illegal EcoRI site found at 1933
    Illegal PstI site found at 1028
    Illegal PstI site found at 1654
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1015
    Illegal EcoRI site found at 1543
    Illegal EcoRI site found at 1711
    Illegal EcoRI site found at 1933
    Illegal PstI site found at 1028
    Illegal PstI site found at 1654
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1015
    Illegal EcoRI site found at 1543
    Illegal EcoRI site found at 1711
    Illegal EcoRI site found at 1933
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1015
    Illegal EcoRI site found at 1543
    Illegal EcoRI site found at 1711
    Illegal EcoRI site found at 1933
    Illegal PstI site found at 1028
    Illegal PstI site found at 1654
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1015
    Illegal EcoRI site found at 1543
    Illegal EcoRI site found at 1711
    Illegal EcoRI site found at 1933
    Illegal PstI site found at 1028
    Illegal PstI site found at 1654
    Illegal AgeI site found at 445
    Illegal AgeI site found at 505
    Illegal AgeI site found at 1234
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1099
    Illegal SapI.rc site found at 1528


Cultivation, Purification and SDS-PAGE

induction condition

To ensure proper folding of mSA and minimize inclusion body formation, we modified the protein buffer by adding biotin. The binding of biotin to mSA can help facilitate proper folding of the Twisted Silinker - GFP protein, reducing the formation of inclusion bodies resulting from misfolding. As a result, we obtained soluble protein extract in the supernatant. The formulation of the buffer and experimental procedures can be found in (protocol) for reference.

We chose 0.1mM IPTG for induction, and the target protein concentration was in a high level, which was the effective induction concentration.

Image Description
Figure 1 | Twisted Silinker - GFP Protein Purification SDS-PAGE gel. Massive expression of Twisted Silinker - GFP protein was performed under 16°C, 0.1mM IPTG induction, followed by nickel column affinity chromatography purification. A Blue Plus V Protein Marker (10-190kDa) was used for size comparison. The lane bands in the image, from left to right, represent the marker, flow-through, the 10mM imidazole elution, the 40mM imidazole elution, the 100mM imidazole elution and the 200mM imidazole elution. The gel was stained with Coomassie brilliant blue and subjected to protein gel analysis.

Purification of Twisted Silinker - GFP

After successfully determining the expression conditions of Twisted Silinker - GFP protein, it is necessary to scale up the culture and proceed with purification. We induced expression with 0.1mM IPTG and allowed it to proceed at 16°C for 18 hours, resulting in a large amount of the target protein. In the process of constructing the expression vector, we incorporated a His-tag into the target protein and utilized a nickel column for affinity chromatography purification based on the specific binding of the His-tagged protein. As shown in Figure 2, the bands displayed successful elution of a significant amount of the target protein using 500mM imidazole solution.















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