Difference between revisions of "Part:BBa K2933198"

 
 
Line 17: Line 17:
 
<partinfo>BBa_K2933198 parameters</partinfo>
 
<partinfo>BBa_K2933198 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
===Usage and Biology===
 +
This composite part is made up with nine basic parts, T7 promoter, the RBS b， the linker h， His tag，the linker a, Sumo tag, linker b, the gene of ARL-1 and T7 terminator.It encodes a protein which is ARL-1 fused with His and Sumo tag. The fusion protein is about 43.0 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of ARL-1 and combine Sumo tag to increased protein solubility. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.<br>
 +
 +
===Molecular cloning===
 +
First, we used the vector pET28B-Sumo to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>
 +
<p style="text-align: center;">
 +
  [[File:ARL-1-PCR.png|500px]]<br>
 +
'''Figure 1.'''  Left: The PCR result of ARL-1. Right: The verification results by enzyme digestion.<br>
 +
</p>
 +
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>

Latest revision as of 13:26, 21 September 2019


T7 promoter+RBS b+linker h+His+Linker a+Sumo+Linker b+ARL-1+T7 terminator

The part consists of T7 promoter,RBS and protein coding(His+Linker a+Sumo+Linker b+ARL-1)and the biological module can be built into E.coil for protein expression.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 390
    Illegal XbaI site found at 47
    Illegal PstI site found at 916
    Illegal PstI site found at 1030
    Illegal PstI site found at 1081
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 390
    Illegal NheI site found at 167
    Illegal NheI site found at 1346
    Illegal PstI site found at 916
    Illegal PstI site found at 1030
    Illegal PstI site found at 1081
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 390
    Illegal BglII site found at 279
    Illegal BamHI site found at 478
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 390
    Illegal XbaI site found at 47
    Illegal PstI site found at 916
    Illegal PstI site found at 1030
    Illegal PstI site found at 1081
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 390
    Illegal XbaI site found at 47
    Illegal PstI site found at 916
    Illegal PstI site found at 1030
    Illegal PstI site found at 1081
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with nine basic parts, T7 promoter, the RBS b， the linker h， His tag，the linker a, Sumo tag, linker b, the gene of ARL-1 and T7 terminator.It encodes a protein which is ARL-1 fused with His and Sumo tag. The fusion protein is about 43.0 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of ARL-1 and combine Sumo tag to increased protein solubility. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pET28B-Sumo to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

ARL-1-PCR.png
Figure 1. Left: The PCR result of ARL-1. Right: The verification results by enzyme digestion.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.