Difference between revisions of "Part:BBa K2302005"
(→Design) |
(→Design) |
||
| Line 6: | Line 6: | ||
===Design=== | ===Design=== | ||
| − | Our team worked to improve the characterization of a signal peptide named PelB,BBa_J32015,a part from the iGEM Registry. This part originated from iGEM2006_Duke team can be used as a signal to allows proteins to be exported outside the cytoplasm. | + | Our team worked to improve the characterization of a signal peptide named PelB, BBa_J32015,a part from the iGEM Registry. This part originated from iGEM2006_Duke team can be used as a signal to allows proteins to be exported outside the cytoplasm. |
To improve the function of this part, we made the codon optimization. To express and secret recombinant CAlB in E. coli, the 5’ terminal of the CALB gene without its own translational initiation codon was combined with the genes coding for the original Pelb signal sequence in Pelb-CALB#1, and Pelb-CALB#2 was combined with improved Pelb signal sequence. The gene of fusion protein was constructed in the pHis vector with tac promoter. | To improve the function of this part, we made the codon optimization. To express and secret recombinant CAlB in E. coli, the 5’ terminal of the CALB gene without its own translational initiation codon was combined with the genes coding for the original Pelb signal sequence in Pelb-CALB#1, and Pelb-CALB#2 was combined with improved Pelb signal sequence. The gene of fusion protein was constructed in the pHis vector with tac promoter. | ||
Revision as of 11:01, 27 October 2017
pelB,a signal peptide that helps the transportation of a gene product out of the membrane.
This sequence encodes a functional signal peptide that can promote the transportation of its fused protein out of the membrane.
Design
Our team worked to improve the characterization of a signal peptide named PelB, BBa_J32015,a part from the iGEM Registry. This part originated from iGEM2006_Duke team can be used as a signal to allows proteins to be exported outside the cytoplasm. To improve the function of this part, we made the codon optimization. To express and secret recombinant CAlB in E. coli, the 5’ terminal of the CALB gene without its own translational initiation codon was combined with the genes coding for the original Pelb signal sequence in Pelb-CALB#1, and Pelb-CALB#2 was combined with improved Pelb signal sequence. The gene of fusion protein was constructed in the pHis vector with tac promoter.
Experiment and Result
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. The predicted molecular weight of fusion protein is different between Pelb-CALB#1 and Pelb-CALB#2, it may be 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.
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 25
Illegal NgoMIV site found at 51 - 1000COMPATIBLE WITH RFC[1000]