Difference between revisions of "Part:BBa K2013021"

Revision as of 15:20, 19 October 2016

pelB-5D

This part contains a special signal peptide that Enhance the secretion of secreted proteins. pelB is a signal peptide promoting protein secretion, through literatures we learn that adding 5 aspartate sequence behind it can enhance its function. Therefore,this part(pelB-5D) is a improvement of existing of BBa_J32015(pelB)

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 54
1000
COMPATIBLE WITH RFC[1000]

Experimental Validation

This part is validated through five ways: amplification， PCR, Enzyme cutting, Sequence and SDS-PAGE.

Amplification

Enzyme:KOD

Primer-F:5′- GAATTCGCGGCCGCTTCTAGATGAAGTACCTGCTGCCGACCG -3′

Primer-R:5′-CCGCTACTAGTAGTCATCGTCATCGTCCGCCAT-3′

Results

PCR

Enzyme:Taq

Primer-F:5′-CCACCTGACGTCTAAGAAAC-3′

Primer-R:5′-GTATTACCGCCTTTGAGTGA-3′

Results

Double digestion

After the assembly ,the plasmid was transferred into the Competent E. coli top10. After culturing overnight in LB,we minipreped the plasmid for double digestion .The first cutting procedure was performed with EcoRI and EcoRV restriction endonuclease. The second cutting procedure was performed with PstI and NcoI restriction endonuclease.The plasmid was cutted in a 25μL system at 37 ℃ for 1 hours. The Electrophoresis was performed on a 1％ Agarose glu

Results

SDS-PAGE

In order to verify the function of pelB-5D, we constructed piGEM2016-001, piGEM2016-002,piGEM2016-module01* and piGEM2016-module01. piGEM2016-001 and piGEM2016-002 are the expression vectors containing pelB-5D and PETase, the only difference between them is the immanent signal peptide of PETase with piGEM2016-002 was removed.piGEM2016-module01 and piGEM2016-module01* is a vector containing PETase and MHETase, the difference between them is that pelB-5D is added in the front of piGEM2016-module01s'MHETase. SDS-PAGE results of piGEM2016-001 and piGEM2016-002 show that the pelB-5D is working.Further more,SDS-PAGE result of piGEM2016-module01 is obviously better than that of piGEM2016-module01*. In short,All results show that pelB-5D can significantly enhance the secretion of extracellular protein.

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 ===SDS-PAGE===
-In order to verify the function of pelB-5D, we constructed piGEM2016-001, piGEM2016-002,piGEM2016-032 and piGEM2016-037. piGEM2016-001 and piGEM2016-002 are the expression vectors containing pelB-5D and PETase, the only difference between them is the immanent signal peptide of PETase with piGEM2016-002  was removed.piGEM2016-037 and piGEM2016-032 is a vector containing PETase and MHETase, the difference between them is that pelB-5D is added in the front of piGEM2016-037s'MHETase. SDS-PAGE results of piGEM2016-001 and piGEM2016-002 show that the pelB-5D is working.Further more,SDS-PAGE result of piGEM2016-037 is obviously better than that of  piGEM2016-032. In short,All results show that pelB-5D can significantly enhance the secretion of extracellular protein.
+In order to verify the function of pelB-5D, we constructed piGEM2016-001, piGEM2016-002,piGEM2016-module01* and piGEM2016-module01. piGEM2016-001 and piGEM2016-002 are the expression vectors containing pelB-5D and PETase, the only difference between them is the immanent signal peptide of PETase with piGEM2016-002  was removed.piGEM2016-module01 and piGEM2016-module01* is a vector containing PETase and MHETase, the difference between them is that pelB-5D is added in the front of piGEM2016-module01s'MHETase. SDS-PAGE results of piGEM2016-001 and piGEM2016-002 show that the pelB-5D is working.Further more,SDS-PAGE result of piGEM2016-module01 is obviously better than that of  piGEM2016-module01*. In short,All results show that pelB-5D can significantly enhance the secretion of extracellular protein.
 https://static.igem.org/mediawiki/2016/0/0b/T--UESTC-China--part21.jpg