Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Binglin Liu
Date created: 2016-10-12 11:00:00
Date modified: 2016-10-12 11:13:51
pelB-5D
| Types | DnaRegion |
| Roles | Tag tag |
| Sequences | BBa_K2013021_sequence (Version 1) |
Description
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)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