Difference between revisions of "Part:BBa K2617017"

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==Characterization==
 
==Characterization==
  
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We have improved the previous part [https://parts.igem.org/Part:BBa_J23100 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.
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We have improved the previous part [https://parts.igem.org/Part:BBa_J23100 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.
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Link to previous part:https://parts.igem.org/Part:BBa_J23100
  
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===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.
 
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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===Experiment Result===
 
===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.
 
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.
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[[File:T--UESTC-China--improvet1.png|500px|center|'''Fig. 1'''The standard curve of pNP.]]
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===Validate the extracellular expression of PETase===
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The activity of the extracelluar fractions was detected by the method of pNPB. And the results were as followed(Fig. 2).
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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.
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[[File:T--UESTC-China--improvet2.png|500px|center|'''Fig. 2 The activity for extracellular fractions of Positive Control and Negative Control]]
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Based on our functional determination test results, the improvement that we did has succeeded. PETase that carrying our improved part (Fig. 2) has much higher activity level than the PETase that carrying promoter BBa_J23100 but without pelB-5D. From our experiment result, we could conclude that our improved part indeed gives the promoter BBa_J23100 the function of extracellular expression.  
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 10:45, 17 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.

Link to previous part:https://parts.igem.org/Part:BBa_J23100

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.

Fig. 1The standard curve of pNP.

The activity of the extracelluar fractions was detected by the method of pNPB. And the results were as followed(Fig. 2).

Fig. 2 The activity for extracellular fractions of Positive Control and Negative Control

Based on our functional determination test results, the improvement that we did has succeeded. PETase that carrying our improved part (Fig. 2) has much higher activity level than the PETase that carrying promoter BBa_J23100 but without pelB-5D. From our experiment result, we could conclude that our improved part indeed gives the promoter BBa_J23100 the function of extracellular expression.