Difference between revisions of "Part:BBa J32015"
Weihongyang (Talk | contribs) (→Improvement) |
Weihongyang (Talk | contribs) (→Improvement) |
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==Improvement== | ==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. | 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. | ||
| + | https://static.igem.org/mediawiki/2016/0/0b/T--UESTC-China--part21.jpg | ||
==Improvement== | ==Improvement== | ||
PelB-CALB#1 is expressing CALB with original signal peptide pelB(about 37kD), pelB-CALB#2 is using improved pelB(about 40 kD). To further demonstrate whether the CALB can secrete to the medium by the signal peptide pelB, the supernatant was directly detected using western blot. 30ug of protein sample was added in every well. It is obvious that the expression of pelB-CALB#2 is largely more than pelB-CALB#1. The different molecular of protein between pelB-CALB#1 and pelB-CALB#2 is may because the protein conformation has been changed after codon optimization. Comparing the expression between A and B, we can know that the funtion of pelB has been impreved | PelB-CALB#1 is expressing CALB with original signal peptide pelB(about 37kD), pelB-CALB#2 is using improved pelB(about 40 kD). To further demonstrate whether the CALB can secrete to the medium by the signal peptide pelB, the supernatant was directly detected using western blot. 30ug of protein sample was added in every well. It is obvious that the expression of pelB-CALB#2 is largely more than pelB-CALB#1. The different molecular of protein between pelB-CALB#1 and pelB-CALB#2 is may because the protein conformation has been changed after codon optimization. Comparing the expression between A and B, we can know that the funtion of pelB has been impreved | ||
remarkablely. So, our improvement is successful. | remarkablely. So, our improvement is successful. | ||
| − | + | ||
Revision as of 13:14, 26 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 54
- 1000COMPATIBLE 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.
Improvement
PelB-CALB#1 is expressing CALB with original signal peptide pelB(about 37kD), pelB-CALB#2 is using improved pelB(about 40 kD). To further demonstrate whether the CALB can secrete to the medium by the signal peptide pelB, the supernatant was directly detected using western blot. 30ug of protein sample was added in every well. It is obvious that the expression of pelB-CALB#2 is largely more than pelB-CALB#1. The different molecular of protein between pelB-CALB#1 and pelB-CALB#2 is may because the protein conformation has been changed after codon optimization. Comparing the expression between A and B, we can know that the funtion of pelB has been impreved remarkablely. So, our improvement is successful.