Difference between revisions of "Part:BBa K2020002"
 
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This composite part consists of the promoter [[Part:BBa_R0010|BBa_R0010]], the ribosome binding site [[Part:BBa_B0034|BBa_B0034]], the newly created BioBrick part [[Part:BBa_K2020001|BBa_K2020001]] and the terminator [[Part:BBa_B0010|BBa_B0010]]. BioBrick BBa_K2020001 is a composite part itself and includes the secretion tag pelB ([[Part:BBa_J32015|BBa_J32015]]) and a subtilisin E gene optimized for ''E. coli'' codon usage ([[Part:BBa_K2020000|BBa_K2020000]]).
 
This composite part consists of the promoter [[Part:BBa_R0010|BBa_R0010]], the ribosome binding site [[Part:BBa_B0034|BBa_B0034]], the newly created BioBrick part [[Part:BBa_K2020001|BBa_K2020001]] and the terminator [[Part:BBa_B0010|BBa_B0010]]. BioBrick BBa_K2020001 is a composite part itself and includes the secretion tag pelB ([[Part:BBa_J32015|BBa_J32015]]) and a subtilisin E gene optimized for ''E. coli'' codon usage ([[Part:BBa_K2020000|BBa_K2020000]]).
  
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For the varification of the function of this part, a skim milk assay on agar plates was performed. Therefore, LB skim milk agar plates containing IPTG and the needed antibiotic were poured and the ''E. coli'' BL21 cells containing the plasmid with the expression system were streaked out.
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For the varification of the function of this part, a skim milk assay on agar plates was performed. Therefore, LB skim milk agar plates containing IPTG and the needed antibiotic were produced and the ''E. coli'' BL21 cells containing the plasmid with the expression system were streaked out.
  
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[[File:T--Aachen--labbook_ecoli_skim_milk_native.png|400px| Skim milk plates assay. Cells containing the empty backbone (left) and cells containing the expression system for native subtilisin E (right) after incubation for 3 days at 30°C.]]
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[[File:T--Aachen--Skim_Milk_plate_1.png|300px| Skim milk plates assay. Cells containing the empty backbone (right) and cells containing the expression system for native subtilisin E (left) after incubation for 3 days at 30°C.]]
  
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The figure shows the '''skim milk plates assay'''. Cells containing the empty backbone (left) and cells containing the expression system for native subtilisin E (right) after incubation for 3 days at 30°C.
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The figure shows the results of a '''skim milk assay'''. This assay utilizes the clearance of skim milk due to proteolysis to detect proteolytic activity. Here, cells containing an empty backbone (right) and cells containing the expression system for native subtilisin E (left) after incubation for 3 days at 30°C can be seen.
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A clearance and therefore a proteolytic activity could only be observed for cells containing the expression system for native subtilisin E. Thus, a proteolytic activity can be deduced. In conclusion, the biobrick BBa_K22020002 causes the expression of native subtilisin E in ''E. coli'' cells.
  
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Comparing the clearance of the skim milk plates, a proteolytic activity could be proven for the cells containing the expression system for native subtilisin E. As a result, these cells growed three days and afterwards, they were able to produce the native protease, which will then digest the skim milk in the agar plates, resulting in a clearance.
 
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In conclusion, subtilisin E in ''E. coli'' was sucessfully expressed and its proteolytic activity were proved via skim milk assay.
 
  
 
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Latest revision as of 13:13, 29 November 2016


expression system for subtilisin E in E. coli

Once introduced into Escherichia coli, this BioBrick is able to produce subtilisin E and simultaneously secret the enzyme into the periplasm of the cell.


Usage and Biology

Subtilisin E is an alkaline serine protease which non-specifically digests proteins. It is naturally produced by Bacillus subtilis.

This composite part consists of the promoter BBa_R0010, the ribosome binding site BBa_B0034, the newly created BioBrick part BBa_K2020001 and the terminator BBa_B0010. BioBrick BBa_K2020001 is a composite part itself and includes the secretion tag pelB (BBa_J32015) and a subtilisin E gene optimized for E. coli codon usage (BBa_K2020000).

For the varification of the function of this part, a skim milk assay on agar plates was performed. Therefore, LB skim milk agar plates containing IPTG and the needed antibiotic were produced and the E. coli BL21 cells containing the plasmid with the expression system were streaked out.

Skim milk plates assay. Cells containing the empty backbone (right) and cells containing the expression system for native subtilisin E (left) after incubation for 3 days at 30°C.

The figure shows the results of a skim milk assay. This assay utilizes the clearance of skim milk due to proteolysis to detect proteolytic activity. Here, cells containing an empty backbone (right) and cells containing the expression system for native subtilisin E (left) after incubation for 3 days at 30°C can be seen.

A clearance and therefore a proteolytic activity could only be observed for cells containing the expression system for native subtilisin E. Thus, a proteolytic activity can be deduced. In conclusion, the biobrick BBa_K22020002 causes the expression of native subtilisin E in E. coli cells.


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 NgoMIV site found at 280
  • 1000
    COMPATIBLE WITH RFC[1000]