Difference between revisions of "Part:BBa K2933137"
(→‎Expression and purification)
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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 11:45, 25 September 2019


His+Linker a+Sumo+Linker b+ARL-1

This part encodes the fusion protein of His tag, sumo tag and ARL-1 to promote the expression and purification of target protein(ARL-1).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 256
    Illegal PstI site found at 782
    Illegal PstI site found at 896
    Illegal PstI site found at 947
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 256
    Illegal NheI site found at 33
    Illegal PstI site found at 782
    Illegal PstI site found at 896
    Illegal PstI site found at 947
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 256
    Illegal BglII site found at 145
    Illegal BamHI site found at 344
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 256
    Illegal PstI site found at 782
    Illegal PstI site found at 896
    Illegal PstI site found at 947
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 256
    Illegal PstI site found at 782
    Illegal PstI site found at 896
    Illegal PstI site found at 947
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with four basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein ARL-1. It encodes a protein which is ARL-1 fused with His-Sumo tag. The fusion protein is about 43.0kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of ARL-1 and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.

Molecular cloning

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.

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.