Difference between revisions of "Part:BBa K2617017"

(→‎Vector design for the validation of expression system)
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===Vector design for the validation of expression system===
 
===Vector design for the validation of expression system===
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For the verification of function, we decided to use the PETase as our reporter protein, which had been used in 2016 iGEM by UESTC-China. Two plasmids were constructed.
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For the function determination, we chose the PETase as our reporter protein because its activity can be easily detected. Thus we constructed two plasmids with and without pelB-5D respectively to compare the function of our improved part and previous part.
  
 
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|<center>1</center>
 
|<center>1</center>
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|<center>Improve-001</center>
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|<center>Positive Control</center>
 
|<center>Kan</center>
 
|<center>Kan</center>
 
|[[File:T--UESTC-China--RBS+pelB+5D+PETase.png|200px|center]]
 
|[[File:T--UESTC-China--RBS+pelB+5D+PETase.png|200px|center]]
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|-
 
|<center>2</center>
 
|<center>2</center>
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|<center>Improve-002</center>
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|<center>Negative Control</center>
 
|<center>Kan</center>
 
|<center>Kan</center>
 
|[[File:T--UESTC-China--RBS+PETase.png|145px|center]]
 
|[[File:T--UESTC-China--RBS+PETase.png|145px|center]]
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Before DNA sequencing, those vectors were verified by restriction enzyme digestion. After electrophoresis analysis, the samples which contained all desired bands were selected and sent for sequencing. The sequencing results showed that all the above constructed vectors were successful.
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===Experiment Result===
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For quantitative assay, a standard curve of pNP ranging from 0-0.8 mM in 100 mM of phosphate buffer (pH 7.4) was measured.
  
 
===Validate the extracellular expression of PETase===
 
===Validate the extracellular expression of PETase===

Revision as of 10:19, 16 October 2018


J23100-RBS-pelB-5D

A combination system of promoter, ribosome binding site and signal peptide for enhancing protein expression(pelB)

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 115
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

We have improved the previous part BBa_J23100 by adding RBS and pelB-5D to make it can be used for extracelluar expression. The combination of promoter, RBS and signal peptide makes it convenient to use as a biobrick and gives it the function of extracellular expression.

Vector design for the validation of expression system

For the function determination, we chose the PETase as our reporter protein because its activity can be easily detected. Thus we constructed two plasmids with and without pelB-5D respectively to compare the function of our improved part and previous part.

No. Vector E.coli resistance Vector map Description
1
Positive Control
Kan
T--UESTC-China--RBS+pelB+5D+PETase.png
BBa_J23100-RBS-pelB+5D-PETase-Ter
2
Negative Control
Kan
T--UESTC-China--RBS+PETase.png
BBa_J23100-RBS-PETase-Ter

Experiment Result

For quantitative assay, a standard curve of pNP ranging from 0-0.8 mM in 100 mM of phosphate buffer (pH 7.4) was measured.

Validate the extracellular expression of PETase

To validate the extracellular expression of PETase, both supernatant and pellets fractions of LB culture were separated by centrifugation(12000rpm,40℃). The results showed that all supernatant fractions collected from all expression systems exhibited higher PETase activity than controls, while the PETase activity in pellet fractions are comparable with the controls, indicating that the extracellular expression system worked, especially for the piGEM2016-001 which showed the highest activity among all tested samples.