Difference between revisions of "Part:BBa K5301017"
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===Characterization===  
 
===Characterization===  
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==PCR==
 
We conducted colony PCR validation to assess the feasibility of the plasmid pathway, as shown in Figure 1, where the corresponding bands are very clear, indicating that the target fragments were successfully introduced into the plasmid and transformed into the bacterial strain.
 
We conducted colony PCR validation to assess the feasibility of the plasmid pathway, as shown in Figure 1, where the corresponding bands are very clear, indicating that the target fragments were successfully introduced into the plasmid and transformed into the bacterial strain.
  
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Figure 1.PCR result of the sGFP1-10 tether.The base sequence length of the sGFP1-10 tether is 1071 base pairs (bp)
 
Figure 1.PCR result of the sGFP1-10 tether.The base sequence length of the sGFP1-10 tether is 1071 base pairs (bp)
 
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==SSDS-PAGE==
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SDS-PAGE was used to verify the purification of the sGFP1-10 tether. Due to the particularity of mSA, we simultaneously purified the soluble proteins from the supernatant and the inclusion body proteins from the pellet after bacterial lysis. It can be observed that the concentration and purity of the purified inclusion body proteins are high, while the concentration of the soluble proteins is low. Further exploration of soluble protein expression or assessment of inclusion body protein activity can be conducted subsequently.
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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0.5;overflow:hidden;">
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https://static.igem.wiki/teams/5301/parts/sgfp1-10-protein.png
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Figure 1.SDS-PAGE of the soluble sGFP1-10 tether.The molecular weight of the sGFP1-10 tether is 39.2kDa.
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==ELISA==
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 07:30, 1 October 2024


sGFP1-10 tether is composed of mSA, a 3C linker, a 6His tag, and sGFP 1-10.

sGFP1-10 tether is composed of mSA, a 3C linker, a 6×His tag, and sGFP 1-10.We construct membrane protein dimers through the interaction between biotin and streptavidin, as well as the self-assembly mechanism of sGFP.

Usage and Biology

mSA is used for binding to biotinylated proteins, and sGFP1-10 and sGFP11 self-assemble to form GFP, thereby creating a protein dimer that can report the outcome with fluorescence.

Characterization

PCR

We conducted colony PCR validation to assess the feasibility of the plasmid pathway, as shown in Figure 1, where the corresponding bands are very clear, indicating that the target fragments were successfully introduced into the plasmid and transformed into the bacterial strain.

sgfp1-10-pcr.png

Figure 1.PCR result of the sGFP1-10 tether.The base sequence length of the sGFP1-10 tether is 1071 base pairs (bp)

SSDS-PAGE

SDS-PAGE was used to verify the purification of the sGFP1-10 tether. Due to the particularity of mSA, we simultaneously purified the soluble proteins from the supernatant and the inclusion body proteins from the pellet after bacterial lysis. It can be observed that the concentration and purity of the purified inclusion body proteins are high, while the concentration of the soluble proteins is low. Further exploration of soluble protein expression or assessment of inclusion body protein activity can be conducted subsequently.

sgfp1-10-protein.png

Figure 1.SDS-PAGE of the soluble sGFP1-10 tether.The molecular weight of the sGFP1-10 tether is 39.2kDa.

ELISA

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 82
    Illegal AgeI site found at 142
  • 1000
    COMPATIBLE WITH RFC[1000]