Difference between revisions of "Part:BBa K2933163"

 
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<partinfo>BBa_K2933163 parameters</partinfo>
 
<partinfo>BBa_K2933163 parameters</partinfo>
 
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===Usage and Biology===
 +
<br>
 +
 +
===Molecular cloning===
 +
First, we used the vector pGEX-6p-1 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;">
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  [[File:TJUSLS China--Elbla2-1-PCR.png]]<br>
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'''Figure 1.'''  Left: The PCR result of Elbla2-1. Right: The verification results by enzyme digestion.<br>
 +
</p>
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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>
 +
 +
===Expression and purification===
 +
'''Pre-expression:'''<br>
 +
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<br>

Revision as of 12:18, 21 September 2019


Tac promoter+RBS a+Linker g+GST+Linker e+ElBlaII

This part consists of Tac promoter,RBS and protein coding sequence (GST+Linker e+ElBla2-1),and the biological module can be built into E.coli for protein expression.


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 1278
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 181


Usage and Biology


Molecular cloning

First, we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

TJUSLS China--Elbla2-1-PCR.png
Figure 1. Left: The PCR result of Elbla2-1. 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.

Expression and purification

Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.