Difference between revisions of "Part:BBa K2933173"

 
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<partinfo>BBa_K2933173 parameters</partinfo>
 
<partinfo>BBa_K2933173 parameters</partinfo>
 
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===Usage and Biology===
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<br>
 +
===Molecular cloning===
 +
 +
First,we obtained BcII by PCR.<br>
 +
<p style="text-align: center;">
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[[File:BcII-194 PCR.jpeg|200px|]]<br>
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'''Figure 1.'''    The PCR result of BcII.<br>
 +
 +
Then 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.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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 +
[[File:BcII-194 6p.jpeg|200px|]]<br>
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'''Figure 2.'''  Left:The plasmid of BcII.Right:The verification results by enzyme digestion.<br>
 +
</p>
 +
===Expression and purification===
 +
'''Exploration of expression condition:'''<br>
 +
Take monoclone in the culture plate into LB tube and cultivate in shaking incubator overnight(10-12h) to activate bacteria.
 +
Test the OD600 number of bacteria, then pipe 5-10ul into each new 5 mL LB tube. Don’t forget to add antibiotic into tubes and mark them.Cultivate in shaking incubator for 3-4 hours until the OD600 of bacteria range from 0.6 to 0.8.Set the gradient of condition to explore how to express it best.We use 0.2mM IPTG, 16°C/0.2mM IPTG, 37°C/0.4mM IPTG, 16°C/0.4mM IPTG, 37°C/0.6mM IPTG, 16°C/0.6mM IPTG, 37°C/0.8mM IPTG, 16°C/0.8mM IPTG, 37°Cas different conditions.<br>
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<p style="text-align: center;">
 +
  [[File:BcII.jpeg|200px|]]<br>
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</p>

Revision as of 12:43, 21 September 2019


Tac promoter+RBS a+Linker g+GST+Linker e+BcII-194

This part consists of Tac promoter,RBS and protein coding sequence (GST+Linker e+BcII-194),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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 181


Usage and Biology


Molecular cloning

First,we obtained BcII by PCR.

BcII-194 PCR.jpeg
Figure 1. The PCR result of BcII.
Then 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.After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.
BcII-194 6p.jpeg
Figure 2. Left:The plasmid of BcII.Right:The verification results by enzyme digestion.

Expression and purification

Exploration of expression condition:
Take monoclone in the culture plate into LB tube and cultivate in shaking incubator overnight(10-12h) to activate bacteria. Test the OD600 number of bacteria, then pipe 5-10ul into each new 5 mL LB tube. Don’t forget to add antibiotic into tubes and mark them.Cultivate in shaking incubator for 3-4 hours until the OD600 of bacteria range from 0.6 to 0.8.Set the gradient of condition to explore how to express it best.We use 0.2mM IPTG, 16°C/0.2mM IPTG, 37°C/0.4mM IPTG, 16°C/0.4mM IPTG, 37°C/0.6mM IPTG, 16°C/0.6mM IPTG, 37°C/0.8mM IPTG, 16°C/0.8mM IPTG, 37°Cas different conditions.

BcII.jpeg