Difference between revisions of "Part:BBa K2933260"
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===Usage and Biology===
 
===Usage and Biology===
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ARL-1 is a type of subclass A beta-lactamases, which is separated from Staphylococcus arlettae. The blaARL-1 was recently discovered and reported, that have a high mutation.<br>
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This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein ARL-1 and T7 terminator. It encodes a protein which is ARL-1 fused with His-Sumo tag. The fusion protein is about 43.0 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.
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Molecular cloning
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===Molecular cloning===
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First, we used the vector pET-28bs to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.
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First, we used the vector pET-28a(+) and the vector pET28a-SUMO to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>
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<p style="text-align: center;">
 
<p style="text-align: center;">
 
   [[File:ARL-1-PCR.png|500px]]<br>
 
   [[File:ARL-1-PCR.png|500px]]<br>
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</p>
 
</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>
 
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>
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===Expression and purification===
 
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'''Pre-expression:'''<br>
 
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The bacteria were cultured in 5mL LB liquid medium with kanamycin(50 μg/mL final concentration) in 37℃ overnight.<br>
 
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'''Massive expressing:'''<br>
 
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After taking samples, we transfered them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 1 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.<br>
 

Revision as of 13:53, 22 September 2019


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

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+ARL-1) and T7 terminator,and the biological module can be build into E.coli for protein expression. This part can be prefaced with promoters of different strengths and types to regulate expression function.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 298
    Illegal PstI site found at 824
    Illegal PstI site found at 938
    Illegal PstI site found at 989
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 298
    Illegal NheI site found at 75
    Illegal NheI site found at 1254
    Illegal PstI site found at 824
    Illegal PstI site found at 938
    Illegal PstI site found at 989
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 298
    Illegal BglII site found at 187
    Illegal BamHI site found at 386
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 298
    Illegal PstI site found at 824
    Illegal PstI site found at 938
    Illegal PstI site found at 989
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 298
    Illegal PstI site found at 824
    Illegal PstI site found at 938
    Illegal PstI site found at 989
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein ARL-1 and T7 terminator. It encodes a protein which is ARL-1 fused with His-Sumo tag. The fusion protein is about 43.0 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein. Molecular cloning First, we used the vector pET-28bs 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.