Difference between revisions of "Part:BBa J32015"

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==Results==
 
==Results==
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To further demonstrate whether the CALB can secrete to the medium by the signal peptide Pelb, the supernatant was directly detected using western blot. CAlB was targeted by His-tag probe. 30ug of protein sample was added in every well. It was obvious that the bands were much more darker in figure B, which indicated the expression of Pelb-CALB#2 was higher than that of Pelb-CALB#1.  
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To further demonstrate whether the CALB can be secreted into the medium by the signal peptide Pelb, the supernatant of culture medium was directly detected using Western blot. Bands with predicted molecular weight of Pelb-CALB in Figure 2B were much stronger than these in Figure 2A with samples using previous Pelb#1 in the iGEM Registry, indicating that the expression of Pelb-CALB#2 was higher than that of Pelb-CALB#1.  
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The predicted molecular weight of fusion protein is different between Pelb-CALB#1 and Pelb-CALB#2, it maybe caused by the changed protein conformation after the codon optimization. Comparing the expression between A and B, it shows that the function of Pelb has been improved remarkablely.
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The predicted molecular weights of fusion proteins Pelb-CALB#1 and Pelb-CALB#2 are somehow slightly different, likely caused alteration in protein conformation after the codon optimization. Comparing the expression between Fig. 2A and 2B, the function of Pelb has been remarkably improved.
  
 
https://static.igem.org/mediawiki/parts/d/d1/NEFU-China_2017_PelB.png
 
https://static.igem.org/mediawiki/parts/d/d1/NEFU-China_2017_PelB.png

Revision as of 01:41, 28 October 2017


PelB leader sequence; directs protein to E. coli periplasmic membrane

The pelB leader sequence is a sequence of amino acids which when attached to a protein, directs the protein to the periplasmic membrane of E. coli, where the sequence is removed by pelB peptidase. It is used to direct coat protein-antigen fusions to the cell surface.

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]


Improvement

When the 5 aspartate repeated sequence（5D） is added to the back of the pelB，its function will be improved. 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.

T--UESTC-China--part21.jpg


Improvement By NEFU-China

To improve the function of this part, we made the codon optimization for its efficient expression in E. coli. To express the recombinant CALB in E. coli and make its secreted, the 5’-terminal of the cDNA without its own translation start codon (ATG) was ligated to our improved Pelb (BBa_K2302005:https://parts.igem.org/Part:BBa_K2302005). The fusion protein was expressed in E. coli BL21 (DE3) using a pHisx6 vector. Then, we determined secreted CALB in the culture medium by Western blot. Compared with the fusion protein expression level in the medium, CALB fused to our improved PelB showed much higher expression than that fused with the peptide in the registry. The use of the Pelb signal sequence for secretory expression is one major improvement for the successful production of our recombinant CALB protein. The Pelb signal enabled the translocation of the CALB into the periplasm, where the protein could be correctly folded with detectable enzyme activity.

Results

To further demonstrate whether the CALB can be secreted into the medium by the signal peptide Pelb, the supernatant of culture medium was directly detected using Western blot. Bands with predicted molecular weight of Pelb-CALB in Figure 2B were much stronger than these in Figure 2A with samples using previous Pelb#1 in the iGEM Registry, indicating that the expression of Pelb-CALB#2 was higher than that of Pelb-CALB#1. The predicted molecular weights of fusion proteins Pelb-CALB#1 and Pelb-CALB#2 are somehow slightly different, likely caused alteration in protein conformation after the codon optimization. Comparing the expression between Fig. 2A and 2B, the function of Pelb has been remarkably improved.

NEFU-China_2017_PelB.png

Link

【1】https://parts.igem.org/Part:BBa_K2302005

【2】http://2017.igem.org/Team:NEFU_China/Basic_Part